Methods for Producing Fabs and IgG Bispecific Antibodies

ABSTRACT

Methods for producing Fabs and IgG bi-specific antibodies comprising expressing nucleic acids encoding designed residues in the CH1/CL interface are provided. Also provided are Fabs and IgG bi-specific antibodies produced according to the provided methods as well as nucleic acids, vectors and host cells encoding the same.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/465,689 filed May 31, 2019, which is the US National Stage ofInternational Application No. PCT/US2017/066296 filed Dec. 14, 2017,which claims the benefit of U.S. Provisional Application No. 62/437,740filed Dec. 22, 2016. The foregoing applications are incorporated hereinby reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML file format and is hereby incorporatedby reference in its entirety. Said XML copy, created on Oct. 10, 2023,is named X20936_44P06WOUS2_SL.xml and is 128,840 bytes in size.

BACKGROUND

Bispecific antibodies integrate the binding properties of two distinctantigen binding proteins into a single molecule. Bispecific antibodiesrepresent an alternative to the co-formulation and/or co-administrationof separate antibody agents or fragments, when the therapeutic targetingof multiple antigens or epitopes is desired. In addition, bispecificantibodies may elicit synergistic activities beyond that of an antibodycombination. For example, bispecific antibodies could be used to bridgedifferent cell types through binding of distinct cell surface receptors,thus facilitating, for example, the targeting of immune cells to tumorcells. Alternately, bispecific antibodies could cross-link and/orcluster separate cell surface signaling receptors to elicit novelmechanisms of action.

The archetypical IgG antibody is a hetero-tetramer comprised of twoidentical heavy chains (HC) and two identical light chains (LC). Each HCassociates with a separate LC to form two identical antigen bindingfragments (Fabs) via assembly interfaces between the HC and LC variabledomains (the V_(H)/V_(L) interface) and the HC constant C_(H)1 and LCconstant domains (the C_(H)1/C_(L) interface.) Once formed, each Fabdirects the binding of the IgG antibody to the same antigenicdeterminant. As a consequence of their modular nature, numerous formatshave been proposed for generating IgG-derived bispecific antibodies.Examples include, diabodies, IgG-single chain variable fragments(IgG-scFv), dual variable domain-IgG (DVD-IgG), Fab-Fab, and IgG-Fab.However, many of these formats alter the native IgG antibody geometryand its resulting stability and solubility, and/or require extensiveengineering to stabilize component variable domains outside of thenative Fab context.

Recently, methods have been described for generating bispecificantibodies retaining the full IgG antibody architecture by co-expressingnucleic acids encoding two distinct HC-LC pairs containing designedmutations in the C_(H)1/C_(L) and/or V_(H)/V_(L) interfaces which, whenexpressed, provide improved assembly of a fully IgG antibody comprisingtwo distinct Fabs. (See Lewis, et al. (2014), Nat. Biotechnol., 32;191-198; and Published PCT Applications WO2014/150973 andWO2014/0154254) In addition, procedures for directing assembly ofparticular HC-HC pairs by introducing modifications into regions of theHC-HC interface to promote improved HC heterodimerization have also beendisclosed in the art (See Leaver-Fay A., et al. (2016), Structure; 24;641-651; Klein et al., mAbs; 4(6); 1-11 (2012); Carter et al., J.Immunol. Methods; 248; 7-15 (2001); Gunasekaran, et al., J. Biol. Chem.;285; 19637-19646 (2010); Zhu et al., Protein Sci.; 6: 781-788 (1997);and Igawa et al., Protein Eng. Des. Sel.; 23; 667-677 (2010)). However,there yet remains a need for alternative methods for generating fullyIgG BsAbs.

SUMMARY

In accordance with the present invention, further methods have beenidentified for achieving assembly of distinct Fabs by co-expressingnucleic acids encoding particular HC-LC pairs which contain designedresidues in the C_(H)1/C_(L) interface. More particularly, the methodsof the present invention achieve improved correct assembly and goodstability of particular Fabs containing kappa LC constant domains (Cκ).Even more particular, the methods of the present invention allow thebinding activities of Fabs of two distinct therapeutic antibodies to becombined in a single fully IgG bi-specific antibody compound. Further,the designs and methods of the present invention may be combined withother known methods for improving HC-LC specific assembly, and/or HC-HCheterodimerization, thus further facilitating assembly of fully IgGBsAbs.

Thus, the present invention provides a method for producing a first andsecond fragment, antigen binding (Fab) comprising: (1) co-expressing ina host cell: (a) a first nucleic acid sequence encoding both a firstheavy chain variable domain and a first human IgG heavy chain constantC_(H)1 domain, wherein said first human IgG heavy chain constant C_(H)1domain comprises an alanine substituted at (or an alanine at) residue145; (b) a second nucleic acid sequence encoding both a first lightchain variable domain and a first human light chain kappa constantdomain, wherein said first human light chain kappa constant domaincomprises an arginine substituted at (or an arginine at) residue 131;(c) a third nucleic acid encoding both a second heavy chain variabledomain and a second human IgG heavy chain constant C_(H)1 domain,wherein said second human IgG heavy chain constant C_(H)1 domain is theWT sequence; and (d) a fourth nucleic acid encoding both a second lightchain variable domain and a second human light chain kappa constantdomain, wherein said second human light chain kappa constant domain isthe WT sequence, wherein each of said first heavy chain and light chainvariable domains comprise three complementarity determining regions(CDRs) which direct binding to a first antigen and further wherein eachof said second heavy chain and light chain variable domains comprisethree complementarity determining regions (CDRs) which direct binding toa second antigen that differs from said first antigen; (2) cultivatingsaid host cell under conditions such that said first and second heavychain variable and human IgG C_(H)1 constant domains and said first andsecond light chain variable and human kappa constant domains areproduced; and (3) recovering from said host cell a first and second Fabwherein said first Fab comprises said first heavy chain variable andhuman IgG constant C_(H)1 domains and said first light chain variableand human kappa constant domains, and said second Fab comprises saidsecond heavy chain variable and human IgG constant C_(H)1 domains andsaid second light chain variable and human kappa constant domains. Moreparticular to this embodiment, the present invention provides a methodwherein said first human IgG heavy chain constant C_(H)1 domain furthercomprises a glutamic acid substituted at (or a glutamic acid at) residue221 and said first human light chain kappa constant domain furthercomprises a lysine substituted at (or a lysine at) residue 123. Evenmore particular to either of the afore-mentioned embodiments, thepresent invention provides a method wherein, said first human IgG heavychain constant C_(H)1 domain further comprises an alanine or glycinesubstituted at (or an alanine or glycine at) residue 188 and said firsthuman light chain kappa constant domain further comprises an isoleucinesubstituted at (or an isoleucine at) residue 176.

As another embodiment, the present invention provides a method forproducing a first and second fragment, antigen binding (Fab) comprising:(1) co-expressing in a host cell: (a) a first nucleic acid sequenceencoding both a first heavy chain variable domain and a first human IgGheavy chain constant C_(H)1 domain, wherein said first human IgG heavychain constant C_(H)1 domain comprises an alanine substituted at (or analanine at) residue 145 and an alanine or glycine substituted at (or analanine or glycine at) residue 188; (b) a second nucleic acid sequenceencoding both a first light chain variable domain and a first humanlight chain kappa constant domain, wherein said first human light chainkappa constant domain comprises an arginine substituted at (or anarginine at) residue 131 and an isoleucine substituted at (or anisoleucine at) residue 176; (c) a third nucleic acid encoding both asecond heavy chain variable domain and a second human IgG heavy chainconstant C_(H)1 domain, wherein said second human IgG heavy chainconstant C_(H)1 domain comprises a glutamic acid substituted at (or aglutamic acid at) residue 221; and (d) a fourth nucleic acid encodingboth a second light chain variable domain and a second human light chainkappa constant domain, wherein said second human light chain kappaconstant domain comprises a lysine substituted at (or a lysine at)residue 123, wherein each of said first heavy chain and light chainvariable domains comprise three complementarity determining regions(CDRs) which direct binding to a first antigen and further wherein eachof said second heavy chain and light chain variable domains comprisethree complementarity determining regions (CDRs) which direct binding toa second antigen that differs from said first antigen; (2) cultivatingsaid host cell under conditions such that said first and second heavychain variable and human IgG C_(H)1 constant domains and said first andsecond light chain variable and human kappa constant domains areproduced; and (3) recovering from said host cell a first and second Fabwherein said first Fab comprises said first heavy chain variable andhuman IgG constant C_(H)1 domains and said first light chain variableand human kappa constant domains, and said second Fab comprises saidsecond heavy chain variable and human IgG constant C_(H)1 domains andsaid second light chain variable and human kappa constant domains.

The C_(H)1/Cκ interface designs of the methods as described above mayalso be combined with designs in V_(H)/V_(L) interface as described inLewis et al. (2014) and WO2014/150973. Thus, as another particularembodiment, the present invention provides any of the afore-mentionedmethods wherein: (a) said first heavy chain variable domain comprises aglutamic acid substituted at (or a glutamic acid at) the residue whichis four amino acids upstream of the first residue of HFR3 according toKabat Numbering and a lysine substituted at (or a lysine at) residue 39;(b) said first light chain variable domain is kappa isotype andcomprises an arginine substituted at (or an arginine at) residue 1 andan aspartic acid substituted at (or an aspartic acid at) residue 38; (c)said second heavy chain variable domain comprises a tyrosine substitutedat (or a tyrosine at) residue 39 and an arginine substituted at (or anarginine at) residue 105; and (d) said second light chain variabledomain is kappa isotype and comprises an arginine substituted at (or anarginine at) residue 38 and an aspartic acid substituted at (or anaspartic acid at) residue 42. Alternately, the present inventionprovides any of the afore-mentioned methods wherein: (a) said firstheavy chain variable domain comprises a tyrosine substituted at (or atyrosine at) residue 39 and an arginine substituted at (or an arginineat) residue 105; (b) said first light chain variable domain is kappaisotype and comprises an arginine substituted at (or an arginine at)residue 38 and an aspartic acid substituted at (or an aspartic acid at)residue 42; (c) said second heavy chain variable domain comprises aglutamic acid substituted at (or a glutamic acid at) the residue whichis four amino acids upstream of the first residue of HFR3 according toKabat Numbering and a lysine substituted at (or a lysine at) residue 39;and (d) said second light chain variable domain is kappa isotype andcomprises an arginine substituted at (or an arginine at) residue 1 andan aspartic acid substituted at (or an aspartic acid at) residue 38.

Even more particular, the present invention provides any of theafore-mentioned methods wherein each of said first and second human IgGheavy chain constant C_(H)1 domains are individually IgG1 or IgG4isotype. The present invention also provides any of the afore-mentionedmethods wherein each of said first and second human IgG heavy chainconstant C_(H)1 domains are IgG1 isotype. The present invention alsoprovides any of the afore-mentioned methods wherein each of said firstand second human IgG heavy chain constant C_(H)1 domains are IgG4isotype.

Further still, the present invention provides any of the afore-mentionedmethods wherein: (a) said first human IgG heavy chain constant C_(H)1domain amino acid sequence is (SEQ ID NO:67), said first human lightchain kappa constant domain amino acid sequence is (SEQ ID NO:57), saidsecond human IgG heavy chain constant C_(H)1 domain amino acid sequenceis (SEQ ID NO: 66), and said second human light chain kappa constantdomain amino acid sequence is (SEQ ID NO:2); or (b) said first human IgGheavy chain constant C_(H)1 domain amino acid sequence is (SEQ IDNO:68), said first human light chain kappa constant domain amino acidsequence is (SEQ ID NO:59), said second human IgG heavy chain constantC_(H)1 domain amino acid sequence is (SEQ ID NO:69), and said secondhuman light chain kappa constant domain amino acid sequence is (SEQ IDNO:61); or (c) said first human IgG heavy chain constant C_(H)1 domainamino acid sequence is (SEQ ID NO:70), said first human light chainkappa constant domain amino acid sequence is (SEQ ID NO:59), said secondhuman IgG heavy chain constant C_(H)1 domain amino acid sequence is (SEQID NO:69), and said second human light chain kappa constant domain aminoacid sequence is (SEQ ID NO:61); or (d) said first human IgG heavy chainconstant C_(H)1 domain amino acid sequence is (SEQ ID NO: 71), saidfirst human light chain kappa constant domain amino acid sequence is(SEQ ID NO: 64), said second human IgG heavy chain constant C_(H)1domain amino acid sequence is (SEQ ID NO:66), and said second humanlight chain kappa constant domain amino acid sequence is (SEQ ID NO:2);or (e) said first human IgG heavy chain constant C_(H)1 domain aminoacid sequence is (SEQ ID NO:72), said first human light chain kappaconstant domain amino acid sequence is (SEQ ID NO: 64), said secondhuman IgG heavy chain constant C_(H)1 domain amino acid sequence is (SEQID NO:66), and said second human light chain kappa constant domain aminoacid sequence is (SEQ ID NO:2).

Further, the present invention provides any of the afore-mentionedmethods wherein each of said first and second light chain variabledomains is human kappa isotype.

The present invention also provides a method for producing an IgGbispecific antibody comprising: (1) co-expressing in a host cell: (a) afirst nucleic acid sequence encoding both a first heavy chain variabledomain and a first human IgG heavy chain constant region, wherein saidfirst human IgG heavy chain constant region comprises a C_(H)1 constantdomain comprising an alanine substituted at (or an alanine at) residue145; (b) a second nucleic acid sequence encoding both a first lightchain variable domain and a first human light chain kappa constantdomain, wherein said first human light chain kappa constant domaincomprises an arginine substituted at (or an arginine at) residue 131;(c) a third nucleic acid encoding both a second heavy chain variabledomain and a second human IgG heavy chain constant region, wherein saidsecond human IgG heavy chain constant region comprises a C_(H)1 constantdomain that is the WT sequence; and (d) a fourth nucleic acid encodingboth a second light chain variable domain and a second human light chainkappa constant domain, wherein said second human light chain kappaconstant domain is the WT sequence, wherein each of said first heavychain and light chain variable domains comprise three complementaritydetermining regions (CDRs) which direct binding to a first antigen andfurther wherein each of said second heavy chain and light chain variabledomains comprise three complementarity determining regions (CDRs) whichdirect binding to a second antigen that differs from said first antigen;(2) cultivating said host cell under conditions such that said first andsecond heavy chain variable and human IgG C_(H)1 constant domains andsaid first and second light chain variable and human kappa constantdomains are produced; and (3) recovering from said host cell an IgGbispecific antibody comprising a first and second fragment, antigenbinding (Fab) wherein said first Fab comprises said first heavy chainvariable and human IgG constant C_(H)1 domains and said first lightchain variable and human kappa constant domains, and said second Fabcomprises said second heavy chain variable and human IgG constant C_(H)1domains and said second light chain variable and human kappa constantdomains. More particular to this embodiment, the present inventionprovides a method for producing an IgG bispecific antibody wherein saidfirst human IgG heavy chain constant C_(H)1 domain further comprises aglutamic acid substituted at (or a glutamic acid at) residue 221 andsaid first human light chain kappa constant domain further comprises alysine substituted at (or a lysine at) residue 123. Even more particularto either of the afore-mentioned embodiments, the present inventionprovides a method for producing an IgG bispecific antibody wherein, saidfirst human IgG heavy chain constant C_(H)1 domain further comprises analanine or glycine substituted at (or an alanine or glycine at) residue188 and said first human light chain kappa constant domain furthercomprises an isoleucine substituted at (or an isoleucine at) residue176.

As another embodiment, the present invention provides a method forproducing an IgG bispecific antibody comprising: (1) co-expressing in ahost cell: (a) a first nucleic acid sequence encoding both a first heavychain variable domain and a first human IgG heavy chain constant region,wherein said first human IgG heavy chain constant region comprises aC_(H)1 constant domain comprising an alanine substituted at (or analanine at) residue 145 and an alanine or glycine substituted at (or analanine or glycine at) residue 188; (b) a second nucleic acid sequenceencoding both a first light chain variable domain and a first humanlight chain kappa constant domain, wherein said first human light chainkappa constant domain comprises an arginine substituted at (or anarginine at) residue 131 and an isoleucine substituted at (or anisoleucine at) residue 176; (c) a third nucleic acid encoding both asecond heavy chain variable domain and a second human IgG heavy chainconstant region, wherein said second IgG heavy chain constant regioncomprises a C_(H)1 domain comprising a glutamic acid substituted at (ora glutamic acid at) residue 221; and (d) a fourth nucleic acid encodingboth a second light chain variable domain and a second human light chainkappa constant domain, wherein said second human light chain kappacomprises a lysine substituted at (or a lysine at) residue 123, whereineach of said first heavy chain and light chain variable domains comprisethree complementarity determining regions (CDRs) which direct binding toa first antigen and further wherein each of said second heavy chain andlight chain variable domains comprise three complementarity determiningregions (CDRs) which direct binding to a second antigen that differsfrom said first antigen; (2) cultivating said host cell under conditionssuch that said first and second heavy chain variable and human IgGC_(H)1 constant domains and said first and second light chain variableand human kappa constant domains are produced; and (3) recovering fromsaid host cell an IgG bispecific antibody comprising a first and secondFab wherein said first Fab comprises said first heavy chain variable andhuman IgG constant C_(H)1 domains and said first light chain variableand human kappa constant domains, and said second Fab comprises saidsecond heavy chain variable and human IgG constant C_(H)1 domains andsaid second light chain variable and human kappa constant domains.

The C_(H)1/Cκ interface designs of the methods for producing an IgGbispecific antibody, as described above, may also be combined withdesigns in V_(H)/V_(L) interface as described in Lewis et al. (2014) andWO2014/150973. Thus, as another embodiment, the present inventionprovides any of the afore-mentioned methods for producing an IgGbispecific antibody wherein: (a) said first heavy chain variable domaincomprises a glutamic acid substituted at (or a glutamic acid at) theresidue which is four amino acids upstream of the first residue of HFR3according to Kabat Numbering and a lysine substituted at (or a lysineat) residue 39; (b) said first light chain variable domain is kappaisotype and comprises an arginine substituted at (or an arginine at)residue 1 and an aspartic acid substituted at (or an aspartic acid at)residue 38; (c) said second heavy chain variable domain comprises atyrosine substituted at (or a tyrosine at) residue 39 and an argininesubstituted at (or an arginine at) residue 105; and (d) said secondlight chain variable domain is kappa isotype and comprises an argininesubstituted at (or an arginine at) residue 38 and an aspartic acidsubstituted at (or an aspartic acid at) residue 42. Alternately, thepresent invention provides any of the afore-mentioned methods forproducing an IgG bispecific antibody wherein: (a) said first heavy chainvariable domain comprises a tyrosine substituted at (or a tyrosine at)residue 39 and an arginine substituted at (or an arginine at) residue105; (b) said first light chain variable domain is kappa isotype andcomprises an arginine substituted at (or an arginine at) residue 38 andan aspartic acid substituted at (or an aspartic acid at) residue 42; (c)said second heavy chain variable domain comprises a glutamic acidsubstituted at (or a glutamic acid at) the residue which is four aminoacids upstream of the first residue of HFR3 according to Kabat Numberingand a lysine substituted at (or a lysine at) residue 39; and (d) saidsecond light chain variable domain is kappa isotype and comprises anarginine substituted at (or an arginine at) residue 1 and an asparticacid substituted at (or an aspartic acid at) residue 38.

The C_(H)1/Cκ interface designs of the methods for producing an IgGbispecific antibody, as described above, may also be combined withdesigns in C_(H)3/C_(H)3 interface as described in Leaver-Fay A., et al.(2016), Structure; 24; 641-651 and WO 2016/118742 A1. Thus, as a furtherparticular embodiment, the present invention provides any of theafore-mentioned methods for producing an IgG bispecific antibodywherein: (a) one of said first or second human IgG constant regionscomprises a C_(H)3 domain comprising an alanine substituted at (or analanine at) residue 407; and the other of said first or second human IgGconstant regions comprises a C_(H)3 domain comprising a valinesubstituted at (or a valine at) residue 366 and a valine substituted at(or a valine at) residue 409; or (b) one of said first or second humanIgG constant regions comprises a C_(H)3 domain comprising an alaninesubstituted at (or an alanine at) residue 407 and a methioninesubstituted at (or a methionine at) residue 399; and the other of saidfirst or second human IgG constant regions comprises a C_(H)3 domaincomprising a valine substituted at (or a valine at) residue 366 and avaline substituted at (or a valine at) residue 409; or (c) one of saidfirst or second human IgG constant regions comprises a C_(H)3 domaincomprising an alanine substituted at (or an alanine at) residue 407, amethionine substituted at (or a methionine at) residue 399, and anaspartic acid substituted at (or an aspartic acid at) residue 360; andthe other of said first or second human IgG constant regions comprises aC_(H)3 domain comprising a valine substituted at (or a valine at)residue 366, a valine substituted at (or a valine at) residue 409, andan arginine substituted at (or an arginine at) residues 345 and 347; or(d) one of said first or second human IgG constant regions comprises aC_(H)3 domain comprising an alanine substituted at (or an alanine at)residue 407, an aspartic acid substituted at (or an aspartic acid at)residue 357, and a glutamine substituted at (or a glutamine at) residue364; and the other of said first or second human IgG constant regionscomprises a C_(H)3 domain comprising a valine substituted at (or avaline at) residue 366, a valine substituted at (or a valine at) residue409, a serine substituted at (or a serine at) residue 349, and atyrosine substituted at (or a tyrosine at) residue 370; or (e) one ofsaid first or second human IgG constant regions comprises a C_(H)3domain comprising an alanine substituted at (or an alanine at) residue407, an aspartic acid substituted at (or an aspartic acid at) residue357, and a glutamine substituted at (or a glutamine at) residue 364; andthe other of said first or second human IgG constant regions comprises aC_(H)3 domain comprising a methionine substituted at (or a methionineat) residue 366, a valine substituted at (or a valine at) residue 409, aserine substituted at (or a serine at) residue 349, and a tyrosinesubstituted at (or a tyrosine at) residue 370; or (f) one of said firstor second human IgG constant regions comprises a C_(H)3 domaincomprising an alanine substituted at (or an alanine at) residue 407, anaspartic acid substituted at (or an aspartic acid at) residue 357, andan arginine substituted at (or an arginine at) residue 364; and theother of said first or second human IgG constant regions comprises aC_(H)3 domain comprising a valine substituted at (or a valine at)residue 366, a valine substituted at (or a valine at) residue 409, aserine substituted at (or a serine at) residue 349, and a tyrosinesubstituted at (or a tyrosine at) residue 370; or (g) one of said firstor second human IgG constant regions comprises a C_(H)3 domaincomprising an alanine substituted at (or an alanine at) residue 407, aglycine substituted at (or a glycine at) residue 356, an aspartic acidsubstituted at (or an aspartic acid at) residue 357, and a glutaminesubstituted at (or a glutamine at) residue 364; and the other of saidfirst or second human IgG constant regions comprises a C_(H)3 domaincomprising a valine substituted at (or a valine at) residue 366, avaline substituted at (or a valine at) residue 409, a serine substitutedat (or a serine at) residue 349, and a tyrosine substituted at (or atyrosine at) residue 370; or (h) one of said first or second human IgGconstant regions comprises a C_(H)3 domain comprising an alaninesubstituted at (or an alanine at) residue 407, a glycine substituted at(or a glycine at) residue 356, an aspartic acid substituted at (or anaspartic acid at) residue 357, and a glutamine substituted at (or aglutamine at) residue 364; and the other of said first or second humanIgG constant regions comprises a C_(H)3 domain comprising a methioninesubstituted at (or a methionine at) residue 366, a valine substituted at(or a valine at) residue 409, a serine substituted at (or a serine at)residue 349, and a tyrosine substituted at (or a tyrosine at) residue370; or (i) one of said first or second human IgG constant regionscomprises a C_(H)3 domain comprising an alanine substituted at (or analanine at) residue 407, an aspartic acid substituted at (or an asparticacid at) residue 357, and an arginine substituted at (or an arginine at)residue 364; and the other of said first or second human IgG constantregions comprises a C_(H)3 domain comprising a methionine substituted at(or a methionine at) residue 366, a valine substituted at (or a valineat) residue 409, a serine substituted at (or a serine at) residue 349,and a tyrosine substituted at (or a tyrosine at) residue 370.

As yet another embodiment, the present invention provides any of theafore-mentioned methods for producing an IgG bispecific antibody whereineach of said first and second human IgG heavy chain constant regions areindividually IgG1 or IgG4 isotype, and more particularly each are IgG1,or each are IgG4.

Further still, the present invention provides any of the afore-mentionedmethods for producing an IgG bispecific antibody wherein: (a) said firsthuman IgG heavy chain constant C_(H)1 domain amino acid sequence is (SEQID NO: 67), said first human light chain kappa constant domain aminoacid sequence is (SEQ ID NO:57), said second human IgG heavy chainconstant C_(H)1 domain amino acid sequence is (SEQ ID NO:66), and saidsecond human light chain kappa constant domain amino acid sequence is(SEQ ID NO:2); or (b) said first human IgG heavy chain constant C_(H)1domain amino acid sequence is (SEQ ID NO:68), said first human lightchain kappa constant domain amino acid sequence is (SEQ ID NO:59), saidsecond human IgG heavy chain constant C_(H)1 domain amino acid sequenceis (SEQ ID NO:69), and said second human light chain kappa constantdomain amino acid sequence is (SEQ ID NO:61); or (c) said first humanIgG heavy chain constant C_(H)1 domain amino acid sequence is (SEQ IDNO:70), said first human light chain kappa constant domain amino acidsequence is (SEQ ID NO: 59), said second human IgG heavy chain constantC_(H)1 domain amino acid sequence is (SEQ ID NO:69), and said secondhuman light chain kappa constant domain amino acid sequence is (SEQ IDNO:61); or (d) said first human IgG heavy chain constant C_(H)1 domainamino acid sequence is (SEQ ID NO:71), said first human light chainkappa constant domain amino acid sequence is (SEQ ID NO: 64), saidsecond human IgG heavy chain constant C_(H)1 domain amino acid sequenceis (SEQ ID NO:66), and said second human light chain kappa constantdomain amino acid sequence is (SEQ ID NO:2); or (e) said first human IgGheavy chain constant C_(H)1 domain amino acid sequence is (SEQ IDNO:72), said first human light chain kappa constant domain amino acidsequence is (SEQ ID NO:64), said second human IgG heavy chain constantC_(H)1 domain amino acid sequence is (SEQ ID NO:66), and said secondhuman light chain kappa constant domain amino acid sequence is (SEQ IDNO:2).

As a further particular embodiment, the present invention provides anyof the afore-mentioned methods for producing an IgG bispecific antibodywherein: (a) one of said first or second human IgG constant regionscomprises an Fc domain amino acid sequence that is (SEQ ID NO:83); andthe other of said first or second human IgG constant regions comprisesan Fc domain amino acid sequence that is (SEQ ID NO:78); or (b) one ofsaid first or second human IgG constant regions comprises an Fc domainamino acid sequence that is (SEQ ID NO:74); and the other of said firstor second human IgG constant regions comprises an Fc domain amino acidsequence that is (SEQ ID NO:78); or (c) one of said first or secondhuman IgG constant regions comprises an Fc domain amino acid sequencethat is (SEQ ID NO:75); and the other of said first or second human IgGconstant regions comprises an Fc domain amino acid sequence that is (SEQID NO:79); or (d) one of said first or second human IgG constant regionscomprises an Fc domain amino acid sequence that is (SEQ ID NO:76); andthe other of said first or second human IgG constant regions comprisesan Fc domain amino acid sequence that is (SEQ ID NO:80); or (e) one ofsaid first or second human IgG constant regions comprises an Fc domainamino acid sequence that is (SEQ ID NO:77); and the other of said firstor second human IgG constant regions comprises an Fc domain amino acidsequence that is (SEQ ID NO:80); or (f) one of said first or secondhuman IgG constant regions comprises an Fc domain amino acid sequencethat is (SEQ ID NO:76); and the other of said first or second human IgGconstant regions comprises an Fc domain amino acid sequence that is (SEQID NO:82); or (g) one of said first or second human IgG constant regionscomprises an Fc domain amino acid sequence that is (SEQ ID NO:76); andthe other of said first or second human IgG constant regions comprisesan Fc domain amino acid sequence that is (SEQ ID NO:81); or (h) one ofsaid first or second human IgG constant regions comprises an Fc domainamino acid sequence that is (SEQ ID NO:77); and the other of said firstor second human IgG constant regions comprises an Fc domain amino acidsequence that is (SEQ ID NO:81); or (i) one of said first or secondhuman IgG constant regions comprises an Fc domain amino acid sequencethat is (SEQ ID NO:77); and the other of said first or second human IgGconstant regions comprises an Fc domain amino acid sequence that is (SEQID NO:82).

Further, the present invention provides any of the afore-mentionedmethods for producing an IgG bispecific antibody wherein each of saidfirst and second light chain variable domains is human kappa isotype.

The present invention also provides an IgG bispecific antibodycomprising: (a) a first heavy chain, wherein said first heavy chaincomprises a first variable domain (V_(H)) and a first human IgG1 or IgG4constant region, wherein said first human IgG1 or IgG4 constant regioncomprises an alanine substituted at (or an alanine at) residue 145 ofthe C_(H)1 domain; (b) a first light chain, wherein said first lightchain comprises a first variable domain (V_(L)) and a first human lightchain kappa constant domain (C_(κ)), wherein said first human lightchain kappa constant domain comprises an arginine substituted at (or anarginine at) residue 131; (c) a second heavy chain, wherein said secondheavy chain comprises a second variable domain (V_(H)) and a secondhuman IgG1 or IgG4 constant region, wherein said second human IgG1 orIgG4 heavy chain constant region comprises a C_(H)1 constant domain thatis the WT sequence; and (d) a second light chain, wherein said secondlight chain comprises a second variable domain (V_(L)) and a secondhuman light chain kappa constant domain (C_(κ)), wherein said secondhuman light chain kappa constant domain is the WT sequence, wherein eachof said first heavy chain and light chain variable domains comprisethree complementarity determining regions (CDRs) which direct binding toa first antigen and further wherein each of said second heavy chain andlight chain variable domains comprise three complementarity determiningregions (CDRs) which direct binding to a second antigen that differsfrom said first antigen. More particular to this embodiment, the presentinvention provides an IgG bispecific antibody wherein said first humanIgG1 or IgG4 heavy chain constant region C_(H)1 domain further comprisesa glutamic acid substituted at (or a glutamic acid at) residue 221 andsaid first human light chain kappa constant domain further comprises alysine substituted at (or a lysine at) residue 123. Even more particularto either of the afore-mentioned embodiments, the present inventionprovides an IgG bispecific antibody wherein said first human IgG1 orIgG4 heavy chain constant region C_(H)1 domain further comprises analanine or glycine substituted at (or an alanine or glycine at) residue188 and said first human light chain kappa constant domain furthercomprises an isoleucine substituted at (or an isoleucine at) residue176.

The present invention also provides an IgG bispecific antibodycomprising: (a) a first heavy chain, wherein said first heavy chaincomprises a first variable domain (V_(H)) and a first human IgG1 or IgG4constant region, wherein said first human IgG1 or IgG4 constant regioncomprises an alanine substituted at (or an alanine at) residue 145 andan alanine or glycine substituted at (or an alanine or glycine at)residue 188 of the C_(H)1 domain; (b) a first light chain, wherein saidfirst light chain comprises a first variable domain (V_(L)) and a firsthuman light chain kappa constant domain (C_(κ)), wherein said firsthuman light chain kappa constant domain comprises an argininesubstituted at (or an arginine at) residue 131 and an isoleucinesubstituted at (or an isoleucine at) residue 176; (c) a second heavychain, wherein said second heavy chain comprises a second variabledomain (V_(H)) and a second human IgG1 or IgG4 constant region, whereinsaid second human IgG1 or IgG4 heavy chain constant region comprises aglutamic acid substituted at (or a glutamic acid at) residue 221 of theC_(H)1 constant domain; and (d) a second light chain, wherein saidsecond light chain comprises a second variable domain (V_(L)) and asecond human light chain kappa constant domain (C_(κ)), wherein saidsecond human light chain kappa constant domain comprises a lysinesubstituted at (or a lysine at) residue 123, wherein each of said firstheavy chain and light chain variable domains comprise threecomplementarity determining regions (CDRs) which direct binding to afirst antigen and further wherein each of said second heavy chain andlight chain variable domains comprise three complementarity determiningregions (CDRs) which direct binding to a second antigen that differsfrom said first antigen.

The C_(H)1/Cκ interface designs of the IgG bispecific antibodies asdescribed above may also be combined with designs in V_(H)/V_(L)interface as described in Lewis et al. (2014) and WO2014/150973. Thus,as another embodiment, the present invention provides any of theafore-mentioned IgG bispecific antibodies wherein: (a) said first heavychain variable domain comprises a glutamic acid substituted at (or aglutamic acid at) the residue which is four amino acids upstream of thefirst residue of HFR3 according to Kabat Numbering and a lysinesubstituted at (or a lysine at) residue 39; (b) said first light chainvariable domain is kappa isotype and comprises an arginine substitutedat (or an arginine at) residue 1 and an aspartic acid substituted at (oran aspartic acid at) residue 38; (c) said second heavy chain variabledomain comprises a tyrosine substituted at (or a tyrosine at) residue 39and an arginine substituted at (or an arginine at) residue 105; and (d)said second light chain variable domain is kappa isotype and comprisesan arginine substituted at (or an arginine at) residue 38 and anaspartic acid substituted at (or an aspartic acid at) residue 42.Alternately, the present invention provides any of the afore-mentionedIgG bispecific antibodies wherein: (a) said first heavy chain variabledomain comprises a tyrosine substituted at (or a tyrosine at) residue 39and an arginine substituted at (or an arginine at) residue 105; (b) saidfirst light chain variable domain is kappa isotype and comprises anarginine substituted at (or an arginine at) residue 38 and an asparticacid substituted at (or an aspartic acid at) residue 42; (c) said secondheavy chain variable domain comprises a glutamic acid substituted at (ora glutamic acid at) the residue which is four amino acids upstream ofthe first residue of HFR3 according to Kabat Numbering and a lysinesubstituted at (or a lysine at) residue 39; and (d) said second lightchain variable domain is kappa isotype and comprises an argininesubstituted at (or an arginine at) residue 1 and an aspartic acidsubstituted at (or an aspartic acid at) residue 38.

The C_(H)1/Cκ interface designs of the IgG bispecific antibodies, asdescribed above, may also be combined with designs in C_(H)3/C_(H)3interface as described in Leaver-Fay A., et al. (2016), Structure; 24;641-651 and WO 2016/118742 A1. Thus, as a further particular embodiment,the present invention provides any of the afore-mentioned IgG bispecificantibodies wherein: (a) one of said first or second human IgG1 or IgG4constant regions comprises a C_(H)3 domain comprising an alaninesubstituted at (or an alanine at) residue 407; and the other of saidfirst or second human IgG1 or IgG4 constant regions comprises a C_(H)3domain comprising a valine substituted at (or a valine at) residue 366and a valine substituted at (or a valine at) residue 409; or (b) one ofsaid first or second human IgG1 or IgG4 constant regions comprises aC_(H)3 domain comprising an alanine substituted at (or an alanine at)residue 407 and a methionine substituted at (or a methionine at) residue399; and the other of said first or second human IgG1 or IgG4 constantregions comprises a C_(H)3 domain comprising a valine substituted at (ora valine at) residue 366 and a valine substituted at (or a valine at)residue 409; or (c) one of said first or second human IgG1 or IgG4constant regions comprises a C_(H)3 domain comprising an alaninesubstituted at (or an alanine at) residue 407, a methionine substitutedat (or a methionine at) residue 399, and an aspartic acid substituted at(or an aspartic acid at) residue 360; and the other of said first orsecond human IgG1 or IgG4 constant regions comprises a C_(H)3 domaincomprising a valine substituted at (or a valine at) residue 366, avaline substituted at (or a valine at) residue 409, and an argininesubstituted at (or an arginine at) residues 345 and 347; or (d) one ofsaid first or second human IgG1 or IgG4 constant regions comprises aC_(H)3 domain comprising an alanine substituted at (or an alanine at)residue 407, an aspartic acid substituted at (or an aspartic acid at)residue 357, and a glutamine substituted at (or a glutamine at) residue364; and the other of said first or second human IgG1 or IgG4 constantregions comprises a C_(H)3 domain comprising a valine substituted at (ora valine at) residue 366, a valine substituted at (or a valine at)residue 409, a serine substituted at (or a serine at) residue 349, and atyrosine substituted at (or a tyrosine at) residue 370; or (e) one ofsaid first or second human IgG1 or IgG4 constant regions comprises aC_(H)3 domain comprising an alanine substituted at (or an alanine at)residue 407, an aspartic acid substituted at (or an aspartic acid at)residue 357, and a glutamine substituted at (or a glutamine at) residue364; and the other of said first or second human IgG1 or IgG4 constantregions comprises a C_(H)3 domain comprising a methionine substituted at(or a methionine at) residue 366, a valine substituted at (or a valineat) residue 409, a serine substituted at (or a serine at) residue 349,and a tyrosine substituted at (or a tyrosine at) residue 370; or (f) oneof said first or second human IgG1 or IgG4 constant regions comprises aC_(H)3 domain comprising an alanine substituted at (or an alanine at)residue 407, an aspartic acid substituted at (or an aspartic acid at)residue 357, and an arginine substituted at (or an arginine at) residue364; and the other of said first or second human IgG1 or IgG4 constantregions comprises a C_(H)3 domain comprising a valine substituted at (ora valine at) residue 366, a valine substituted at (or a valine at)residue 409, a serine substituted at (or a serine at) residue 349, and atyrosine substituted at (or a tyrosine at) residue 370; or (g) one ofsaid first or second human IgG1 or IgG4 constant regions comprises aC_(H)3 domain comprising an alanine substituted at (or an alanine at)residue 407, a glycine substituted at (or a glycine at) residue 356, anaspartic acid substituted at (or an aspartic acid at) residue 357, and aglutamine substituted at (or a glutamine at) residue 364; and the otherof said first or second human IgG1 or IgG4 constant regions comprises aC_(H)3 domain comprising a valine substituted at (or a valine at)residue 366, a valine substituted at (or a valine at) residue 409, aserine substituted at (or a serine at) residue 349, and a tyrosinesubstituted at (or a tyrosine at) residue 370; or (h) one of said firstor second human IgG1 or IgG4 constant regions comprises a C_(H)3 domaincomprising an alanine substituted at (or an alanine at) residue 407, aglycine substituted at (or a glycine at) residue 356, an aspartic acidsubstituted at (or an aspartic acid at) residue 357, and a glutaminesubstituted at (or a glutamine at) residue 364; and the other of saidfirst or second human IgG1 or IgG4 constant regions comprises a C_(H)3domain comprising a methionine substituted at (or a methionine at)residue 366, a valine substituted at (or a valine at) residue 409, aserine substituted at (or a serine at) residue 349, and a tyrosinesubstituted at (or a tyrosine at) residue 370; or (i) one of said firstor second human IgG1 or IgG4 constant regions comprises a C_(H)3 domaincomprising an alanine substituted at (or an alanine at) residue 407, anaspartic acid substituted at (or an aspartic acid at) residue 357, andan arginine substituted at (or an arginine at) residue 364; and theother of said first or second human IgG1 or IgG4 constant regionscomprises a C_(H)3 domain comprising a methionine substituted at (or amethionine at) residue 366, a valine substituted at (or a valine at)residue 409, a serine substituted at (or a serine at) residue 349, and atyrosine substituted at (or a tyrosine at) residue 370.

Even more particular, the present invention provides any of theafore-mentioned IgG bispecific antibodies wherein one of said first orsecond human IgG1 or IgG4 heavy chain constant regions is IgG1 isotypeand the other of said first or second human IgG1 or IgG4 heavy chainconstant regions is IgG4 isotype. The present invention also providesany of the afore-mentioned IgG bispecific antibodies wherein each ofsaid first and second human IgG1 or IgG4 heavy chain constant regionsare IgG1 isotype. The present invention also provides any of theafore-mentioned IgG bispecific antibodies wherein each of said first andsecond human IgG1 or IgG4 heavy chain constant regions are IgG4 isotype.

Further still, the present invention provides any of the afore-mentionedIgG bispecific antibodies wherein: (a) said first human IgG heavy chainconstant region comprises a C_(H)1 domain amino acid sequence that is(SEQ ID NO:67), said first human light chain kappa constant domain aminoacid sequence is (SEQ ID NO:57), said second human IgG heavy chainconstant region comprises a C_(H)1 domain amino acid sequence that is(SEQ ID NO:66), and said second human light chain kappa constant domainamino acid sequence is (SEQ ID NO:2); or (b) said first human IgG heavychain constant region comprises a C_(H)1 domain amino acid sequence thatis (SEQ ID NO:68), said first human light chain kappa constant domainamino acid sequence is (SEQ ID NO:59), said second human IgG heavy chainconstant region comprises a C_(H)1 domain amino acid sequence that is(SEQ ID NO:69), and said second human light chain kappa constant domainamino acid sequence is (SEQ ID NO:61); or (c) said first human IgG heavychain constant region comprises a C_(H)1 domain amino acid sequence thatis (SEQ ID NO:70), said first human light chain kappa constant domainamino acid sequence is (SEQ ID NO:59), said second human IgG heavy chainconstant region comprises a C_(H)1 domain amino acid sequence that is(SEQ ID NO:69), and said second human light chain kappa constant domainamino acid sequence is (SEQ ID NO:61); or (d) said first human IgG heavychain constant region comprises a C_(H)1 domain amino acid sequence thatis (SEQ ID NO:71), said first human light chain kappa constant domainamino acid sequence is (SEQ ID NO:64), said second human IgG heavy chainconstant region comprises a C_(H)1 domain amino acid sequence that is(SEQ ID NO: 66), and said second human light chain kappa constant domainamino acid sequence is (SEQ ID NO:2); or (e) said first human IgG heavychain constant region comprises a C_(H)1 domain amino acid sequence thatis (SEQ ID NO:72), said first human light chain kappa constant domainamino acid sequence is (SEQ ID NO:64), said second human IgG heavy chainconstant region comprises a C_(H)1 domain amino acid sequence that is(SEQ ID NO:66), and said second human light chain kappa constant domainamino acid sequence is (SEQ ID NO:2).

As a further particular embodiment, the present invention provides anyof the afore-mentioned IgG bispecific antibodies wherein: (a) one ofsaid first or second human IgG constant regions comprises an Fe domainamino acid sequence that is (SEQ ID NO:83); and the other of said firstor second human IgG constant regions comprises an Fc domain amino acidsequence that is (SEQ ID NO:78); or (b) one of said first or secondhuman IgG constant regions comprises an Fc domain amino acid sequencethat is (SEQ ID NO:74); and the other of said first or second human IgGconstant regions comprises an Fc domain amino acid sequence that is (SEQID NO:78); or (c) one of said first or second human IgG constant regionscomprises an Fc domain amino acid sequence that is (SEQ ID NO:75); andthe other of said first or second human IgG constant regions comprisesan Fc domain amino acid sequence that is (SEQ ID NO:79); or (d) one ofsaid first or second human IgG constant regions comprises an Fc domainamino acid sequence that is (SEQ ID NO:76); and the other of said firstor second human IgG constant regions comprises an Fc domain amino acidsequence that is (SEQ ID NO:80); or (e) one of said first or secondhuman IgG constant regions comprises an Fc domain amino acid sequencethat is (SEQ ID NO:77); and the other of said first or second human IgGconstant regions comprises an Fc domain amino acid sequence that is (SEQID NO:80); or (f) one of said first or second human IgG constant regionscomprises an Fc domain amino acid sequence that is (SEQ ID NO:76); andthe other of said first or second human IgG constant regions comprisesan Fc domain amino acid sequence that is (SEQ ID NO:82); or (g) one ofsaid first or second human IgG constant regions comprises an Fc domainamino acid sequence that is (SEQ ID NO:76); and the other of said firstor second human IgG constant regions comprises an Fc domain amino acidsequence that is (SEQ ID NO:81); or (h) one of said first or secondhuman IgG constant regions comprises an Fc domain amino acid sequencethat is (SEQ ID NO:77); and the other of said first or second human IgGconstant regions comprises an Fc domain amino acid sequence that is (SEQID NO:81); or (i) one of said first or second human IgG constant regionscomprises an Fc domain amino acid sequence that is (SEQ ID NO:77); andthe other of said first or second human IgG constant regions comprisesan Fc domain amino acid sequence that is (SEQ ID NO:82).

Further, the present invention provides any of the afore-mentioned IgGbispecific antibodies wherein each of said first and second light chainvariable domains is human kappa isotype.

The present invention further provides a first and second Fab, or an IgGbispecific antibody produced accord to any one of the processes of thepresent invention. In addition to the preparation of Fabs and fully IgGBsAbs, the methods described herein may also be employed in thepreparation of other multi-valent antigen binding compounds. FIG. 1 ,included herein, provides a schematic diagram of a Fully IgG BsAb, aswell as other antigen binding compounds that one of skill in the artcould prepare using the C_(H)1/Cκ domain designs, or the C_(H)1/Cκdomain designs plus Fab designs, or the C_(H)1/Cκ domain designs plusFab and C_(H)3 designs as described herein.

The present invention further provides nucleic acid sequences encodingthe first and second heavy chains and the first and second light chainsof any of the Fabs or IgG BsAbs of the present invention. In addition,the present invention also provides vectors comprising nucleic acidsequences encoding the first heavy chain, the first light chain, thesecond heavy chain and/or the second light chain of any of the Fabs orIgG BsAbs of the present invention. Further still, the present inventionprovides host cells comprising nucleic acid sequences encoding the firstheavy chain, the first light chain, the second heavy chain and thesecond light chain of any of the Fabs or IgG BsAbs of the presentinvention.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 provides a schematic diagram of antigen binding compounds thatmay be prepared using the C_(H)1/Cκ domain designs, methods orprocedures of the present invention. FIG. 1A provides full IgGbispecific antibody comprising a HC and LC from a first mAb (denotedmAb1) targeting one antigen and a HC and LC from a second mAb (denotedmAb2) recognizing a different antigen. FIG. 1B provides Fab-Fabconstruct comprising a Fab from first mAb (mAb1) and a Fab from secondmAb (mAb2). FIG. 1C provides alternate bispecific constructs comprisinga first mAb (mAb1) and Fab fragments from a second mAb (mAb2).

DETAILED DESCRIPTION

The general structure of an “IgG antibody” is very well-known. A wildtype (WT) antibody of the IgG type is hetero-tetramer of fourpolypeptide chains (two identical “heavy” chains and two identical“light” chains) that are cross-linked via intra- and inter-chaindisulfide bonds. Each heavy chain (HC) is comprised of an N-terminalheavy chain variable region (“V_(H)”) and a heavy chain constant region.The heavy chain constant region is comprised of three domains (C_(H)1,C_(H)2, and C_(H)3) as well as a hinge region (“hinge”) between theC_(H)1 and C_(H)2 domains. Each light chain (LC) is comprised of anN-terminal light chain variable region (“V_(L)”) and a light chainconstant region (“C_(L)”). The V_(L) and C_(L) regions may be of thekappa (“κ”) or lambda (“λ”) isotypes (“Cκ” or “Cλ”, respectively). Eachheavy chain associates with one light chain via interfaces between theheavy chain and light chain variable domains (the V_(H)/V_(L) interface)and the heavy chain constant C_(H)1 and light chain constant domains(the C_(H)1/C_(L) interface). The association between each of theV_(H)-C_(H)1 and V_(L)-C_(L) segments forms two identical antigenbinding fragments (Fabs) which direct antibody binding to the sameantigen or antigenic determinant. Each heavy chain associates with theother heavy chain via interfaces between the hinge-C_(H)2-C_(H)3segments of each heavy chain, with the association between the twoC_(H)2-C_(H)3 segments forming the Fc region of the antibody. Together,each Fab and the Fc form the characteristic “Y-shaped” architecture ofIgG antibodies, with each Fab representing the “arms” of the “Y.” IgGantibodies can be further divided into subtypes, e.g., IgG1, IgG2, IgG3,and IgG4 which differ by the length of the hinge regions, the number andlocation of inter- and intra-chain disulfide bonds and the amino acidsequences of the respective HC constant regions.

The variable regions of each heavy chain-light chain pair associate toform binding sites. The heavy chain variable region (V_(H)) and thelight chain variable region (V_(L)) can be subdivided into regions ofhypervariability, termed complementarity determining regions (“CDRs”),interspersed with regions that are more conserved, termed frameworkregions (“FR”). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. CDRs of the heavy chain maybe referred to as “CDRH1, CDRH2, and CDRH3” and the 3 CDRs of the lightchain may be referred to as “CDRL1, CDRL2 and CDRL3.” The FRs of theheavy chain may be referred to as HFR1, HFR2, HFR3 and HFR4 whereas theFRs of the light chain may be referred to as LFR1, LFR2, LFR3 and LFR4.The CDRs contain most of the residues which form specific interactionswith the antigen. As used herein, “antigen” or “antigenic determinant”refers to a target protein to which an IgG antibody binds, or to aparticular epitope (on a target protein) to which an IgG antibody binds.

As used herein, the terms “IgG bispecific antibody”, “IgG BsAb”, “fullyIgG bispecific antibody” or “fully IgG BsAb” refer to an antibody of thetypical IgG architecture comprising two distinct Fabs, each of whichdirect binding to a separate antigen or antigenic determinant (i.e.,different target proteins or different epitopes on the same targetprotein), and composed of two distinct IgG heavy chains and two distinctlight chains. The V_(H)-C_(H)1 segment of one heavy chain associateswith the V_(L)-C_(L) segment of one light chain to form a “first” Fab,wherein the V_(H) and V_(L) domains each comprise 3 CDRs which directbinding to a first antigen. The V_(H)-C_(H)1 segment of the second heavychain associates with the V_(L)-C_(L) segment of the second light chainto form a “second” Fab, wherein the V_(H) and V_(L) domains eachcomprise 3 CDRs which direct binding to a second antigen that isdifferent than the first. More particularly, the terms “IgG bispecificantibody”, “IgG BsAb”, “fully IgG bispecific antibody” or “fully IgGBsAb” refer to antibodies wherein the HC constant regions are composedof C_(H)1, C_(H)2, and C_(H)3 domains of the IgG1, IgG2 or IgG4 subtype,and particularly the human IgG1, human IgG2 or human IgG4. Even moreparticular, the terms refer to antibodies wherein the HC constantregions are composed of C_(H)1, C_(H)2, and C_(H)3 domains of the IgG1or IgG4 subtype, and most particularly the human IgG1 or human IgG4subtype. In addition, as used herein, the terms “IgG bispecificantibody”, “IgG BsAb”, “fully IgG bispecific antibody” and “fully IgGBsAb” refer to an antibody wherein the constant regions of eachindividual HC of the antibody are all of the same subtype (for example,each of the C_(H)1, C_(H)2, and C_(H)3 domains of a HC are all of thehuman IgG1 subtype, or each of the C_(H)1, C_(H)2, and C_(H)3 domains ofa HC are all of the IgG2 subtype, or each of the C_(H)1, C_(H)2, andC_(H)3 domains of a HC are all of the IgG4 subtype.) Even moreparticular, the term refers to an antibody wherein the constant regionsof both HCs are all of the same subtype (for example, both HCs haveC_(H)1, C_(H)2, and C_(H)3 domains of the human IgG1 subtype, or bothHCs have C_(H)1, C_(H)2, and C_(H)3 domains of the human IgG2 subtype,or both HCs have C_(H)1, C_(H)2, and C_(H)3 domains of the human IgG4subtype.)

The processes and compounds of the present invention comprise designedamino acid modifications at particular residues within the constant andvariable regions of heavy chain and light chain polypeptides. As one ofordinary skill in the art will appreciate, various numbering conventionsmay be employed for designating particular amino acid residues withinIgG constant and variable region sequences. Commonly used numberingconventions include the “Kabat Numbering” and “EU Index Numbering”systems. “Kabat Numbering” or “Kabat Numbering system”, as used herein,refers to the numbering system devised and set forth by the authors inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed,Public Health Service, National Institutes of Health, Bethesda, MD(1991) for designating amino acid residues in both variable and constantdomains of antibody heavy chains and light chains. “EU Index Numbering”or “EU Index Numbering system”, as used herein, refers to the numberingconvention for designating amino acid residues in antibody heavy chainconstant domains, and is also set forth in Kabat et al. (1991). Otherconventions that include corrections or alternate numbering systems forvariable domains include Chothia (Chothia C, Lesk A M (1987), J Mol Biol196: 901-917; Chothia, et al. (1989), Nature 342: 877-883), IMGT(Lefranc, et al. (2003), Dev Comp Immunol 27: 55-77), and AHo (HoneggerA, Pluckthun A (2001) J Mol Biol 309: 657-670). These references provideamino acid sequence numbering schemes for immunoglobulin variableregions that define the location of variable region amino acid residuesof antibody sequences. Unless otherwise expressly stated herein, allreferences to immunoglobulin heavy chain variable region (i.e., V_(H)),constant region C_(H)1 and hinge amino acid residues (i.e. numbers)appearing in the Examples and Claims are based on the Kabat Numberingsystem, as are all references to the light chain V_(L) and C_(L)residues. All references to immunoglobulin heavy chain constant regionC_(H)2 and C_(H)3 residues (i.e., numbers) are based on the EU IndexNumbering system. Thus, as used herein, the phrase “(according to KabatNumbering)” indicates that the recited amino acid residue number (orposition) is numbered in accordance with the Kabat Numbering system,whereas the phrase “(according to EU Index Numbering)” indicates thatthe recited amino acid residue number (or position) is numbered inaccordance with the EU Index Numbering system. With knowledge of theresidue number according to Kabat Numbering or EU Index Numbering, oneof ordinary skill can apply the teachings of the art to identify aminoacid sequence modifications within the present invention, according toany commonly used numbering convention. Note, while the Examples andClaims of the present invention employ Kabat Numbering or EU IndexNumbering to identify particular amino acid residues, it is understoodthat the SEQ ID NOs appearing in the Examples and Sequence Listingaccompanying the present application, as generated by Patent In Version3.5, provide sequential numbering of amino acids within a givenpolypeptide and, thus, do not conform to the corresponding amino acidresidue numbers as provided by Kabat Numbering or EU Index Numbering.

However, as one of skill in the art will also appreciate, CDR sequencelength may vary between individual IgG molecules and, further, thenumbering of individual residues within a CDR may vary depending on thenumbering convention applied. Thus, to reduce ambiguity in thedesignation of amino acid residues within CDRs, such amino acid residuesmay be identified by first employing Kabat Numbering to identify theN-terminal (first) amino acid residue of a reference FR (e.g., HFR3).The residue comprising the recited amino acid of the designs may then bedenoted as being a fixed number of residues upstream (i.e. in theN-terminal direction) from the first amino acid residue in the referenceFR (e.g., HFR3). For example, a Fab design used in combination with theC_(H)1/Cκ domain designs of the present invention comprises theplacement of a glutamic acid (E) in HCDR2 of a particular HC (i.e., FabDesign “AB” comprising glutamic acid at residue 62 (Kabat Numbering) asdescribed in Lewis, et al. (2014)). The recited glutamic acid is locatedat the residue position four amino acids upstream of the first aminoacid of HFR3, as determined according to Kabat Numbering. In the KabatNumbering system, amino acid residue X66 is the most N-terminal (first)amino acid residue of variable region heavy chain framework three(HFR3). One of ordinary skill can employ such a strategy to identify thefirst amino acid residue (most N-terminal) of heavy chain frameworkthree (HFR3) from any human IgG1, IgG2 or IgG4 antibody variable region.Once this landmark is identified, one can then locate the amino acidresidue four residues upstream (N-terminal) to this location and replacethat amino acid (using standard insertion/deletion methods) with aglutamic acid (E) to achieve the design of the invention. Thus, givenany parental immunoglobulin heavy chain amino acid query sequence ofinterest to use in the processes of the invention, one of ordinary skillin the art of antibody engineering would be able to locate theN-terminal HFR3 residue (according to Kabat Numbering) in said querysequence and then count four amino acid residues upstream therefrom toarrive at the location in HCDR2 that should be a glutamic acid (E).

As used herein, the phrase “ . . . a/an [amino acid name] substituted atresidue . . . ”, in reference to a heavy chain or light chainpolypeptide, refers to substitution of the parental amino acid with theindicated amino acid. By way of example, a heavy chain comprising “alysine substituted at residue 39” refers to a heavy chain wherein theparental amino acid sequence has been mutated to contain a lysine atresidue number 39 in place of the parental amino acid. Such mutationsmay also be represented by denoting a particular amino acid residuenumber, preceded by the parental amino acid and followed by thereplacement amino acid. For example, “Q39K” refers to a replacement of aglutamine at residue 39 with a lysine. Similarly, “39K” refers toreplacement of a parental amino acid with a lysine. One of skill in theart will appreciate, however, that as a result of the interface designmodifications of the present invention, Fab pairs and fully IgG BsAbs(and processes for their preparation) are therefore provided wherein thecomponent HC and LC amino acid sequences comprise the resulting or“replacement” amino acid at the designated residue. Thus, for example, aheavy chain which “comprises a lysine substituted at residue 39” mayalternatively be denoted simply as a heavy chain which “comprises alysine at residue 39.”

As used herein, the phrase “WT” or “WT sequence”, in reference to a HCor LC amino acid residue or polypeptide chain, refers to the wild-typeor native amino acid or sequence of amino acids that naturally occupiesthe residue or residues of the polypeptide chain indicated.

Preferably, an IgG BsAb (or Fab pair) of the present invention exists ina homogeneous or substantially homogeneous population. In an embodiment,the IgG BsAb, Fab, or a nucleic acid encoding a component polypeptidesequence of the IgG BsAb or Fab, is provided in “isolated” form. As usedherein, the term “isolated” refers to a protein, polypeptide or nucleicacid which is free or substantially free from other macromolecularspecies found in a cellular environment.

An IgG BsAb or Fab pair of the present invention can be produced usingtechniques well known in the art, such as recombinant expression inmammalian or yeast cells. In particular, the methods and procedures ofthe Examples herein may be readily employed. In addition, the IgG BsAbsor Fabs of the present invention may be further engineered to compriseframework regions derived from fully human frameworks. A variety ofdifferent human framework sequences may be used in carrying outembodiments of the present invention. As a particular embodiment, theframework regions employed in the processes, as well as the IgG BsAbsand Fab pairs of the present invention are of human origin or aresubstantially human (at least 95%, 97% or 99% of human origin.) Thesequences of framework regions of human origin are known in the art andmay be obtained from The Immunoglobulin Factsbook, by Marie-PauleLefranc, Gerard Lefranc, Academic Press 2001, ISBN 012441351.

Expression vectors capable of directing expression of genes to whichthey are operably linked are well known in the art. Expression vectorscontain appropriate control sequences such as promoter sequences andreplication initiation sites. They may also encode suitable selectionmarkers as well as signal peptides that facilitate secretion of thedesired polypeptide product(s) from a host cell. The signal peptide canbe an immunoglobulin signal peptide or a heterologous signal peptide.Nucleic acids encoding desired polypeptides, for example the componentsof the IgG BsAbs of Fabs prepared according to the processes of thepresent invention, may be expressed independently using differentpromoters to which they are operably linked in a single vector or,alternatively, the nucleic acids encoding the desired products may beexpressed independently using different promoters to which they areoperably linked in separate vectors. In addition, nucleic acids encodinga particular HC/LC pair of the IgG BsAbs or Fabs of the presentinvention may expressed from a first vector, while the other HC/LC pairis expressed from a second vector. Single expression vectors encodingboth HC and both LC components of the IgG BsAbs and Fabs of the presentinvention may be prepared using standard methods. By way of example, apE vector encoding a particular HC/LC pair may be engineered to containa NaeI site 5 prime of a unique SalI site, outside of the HC/LCexpression cassette. The vector may then be modified to contain an AscIsite 5 prime of the SalI site using standard techniques. For example,the NaeI to SalI region may be PCR amplified using a 3′ primercontaining the AscI site adjacent to the SalI site, and the resultingfragment cloned into the recipient pE vector. The expression cassetteencoding a second HC/LC pair, may then be isolated from a second (donor)vector by digesting the vector at suitable restriction sites. Forexample, the donor vector may be engineered with MluI and SalI sites topermit isolation of the second expression cassette. This cassette maythen be ligated into the recipient vector previously digested at theAscI and SalI sites (as AscI and MluI restriction sites have the sameoverlapping ends.)

As used herein, a “host cell” refers to a cell that is stably ortransiently transfected, transformed, transduced or infected withnucleotide sequences encoding a desired polypeptide product or products.Creation and isolation of host cell lines producing an IgG BsAb or Fabpair of the present invention can be accomplished using standardtechniques known in the art.

Mammalian cells are preferred host cells for expression of the IgG BsAband Fab compounds according to the present invention. Particularmammalian cells include HEK293, NS0, DG-44, and CHO cells. Preferably,assembled proteins are secreted into the medium in which the host cellsare cultured, from which the proteins can be recovered and isolated.Medium into which a protein has been secreted may be purified byconventional techniques. For example, the medium may be applied to andeluted from a Protein A or G column using conventional methods. Solubleaggregate and multimers may be effectively removed by common techniques,including size exclusion, hydrophobic interaction, ion exchange,hydroxyapatite or mixed modal chromatography. Recovered products may beimmediately frozen, for example at −70° C., or may be lyophilized. Asone of skill in the art will appreciate, when expressed in certainbiological systems, e.g. mammalian cell lines, antibodies areglycosylated in the Fc region unless mutations are introduced in the Fcto reduce glycosylation. In addition, antibodies may be glycosylated atother positions as well.

The object of the present invention is to provide orthogonal interfaceswhich promote the correct pairing of particular heavy chain Fabfragments (denoted, for example as HC_“A” and HC_“B”) with their cognatelight chain Fab fragments (i.e., LC_“a” and LC_“b”) by introducingparticular mutations into heavy chain C_(H)1/light chain C_(κ) domainpairs. As a result of the present invention, increased correct assemblyof “Aa” and “Bb” dimers is achieved, relative to the incorrectlyassembled “Ab” or “Ba” dimers, when the individual heavy chain and lightchain monomers (i.e., “A”, “B”, “a” and “b” chains) are concomitantlyexpressed in a host cell. The following Examples further illustrate theinvention and provide typical methods and procedures for carrying outvarious particular embodiments of the present invention. However, it isunderstood that the Examples are set forth the by way of illustrationand not limitation, and that various modifications may be made by one ofordinary skill in the art.

Example 1: Computational and Rational Design of Modifications ofC_(H)1/Cκ Interface

The identification and design of the particular C_(H)1/C_(κ) residuemutations resulted from an iterative combination of computational andrational approaches. Starting from the high resolution crystal structureof the human IgG1 C_(H)1/CG interface (PDB ID 4NZU from which variabledomains are removed) heavy chain residues 122, 124, 139, 141, 143, 145,174, 188, and 190 and light chain residues 116, 118, 124, 131, 133, 135,and 176 (all according to Kabat Numbering) are selected for initialmutation, allowing substitution to all amino acids except cysteine.Using the Rosetta multistate design (MSD) protocol, and related modelingapplications, the resulting sequences are imposed on each of the fourdimer and four monomer species to computationally identify potentialmodifications that favor correct “Aa” and “Bb” dimer formation.

Briefly, the MSD protocol in Rosetta explores sequence space, and foreach sequence, calculates an energy for each of several “states”treating phenomena such as van der Waals force and hydrogen bondingforces. The “states” represent different combinations and/orconformations of the various species being modeled. MSD optimizes thesequence side chain conformations, without changing backboneconformations, and aggregates the energies into a fitness metric thatreflects how well particular sequences meet design objectives. Thefitness values then guide the search through sequence space, andsequences with improved fitness scores can be identified from thesimulation. To facilitate the present designs, a fitness function ischosen that favors the binding energies of the correctly paired “Aa” and“Bb” dimers, while disfavoring the binding energies of the mis-paired“Ab” and “Ba” dimers.

To remove an artifact of the fixed-backbone simulation, the weight givento the destabilization of the mis-paired interactions is capped in thefitness function employed in the present methods. The fixed-backbonesimulation has a limitation in that it blinds MSD to the possibilitythat collisions introduced across the interface for mis-pairedinteractions can be resolved by moving the backbone slightly. Suchslight motions can be approximated, however, by using a repertoire ofalternate conformations for the mis-paired interactions. A repertoire ofalternate backbone conformations is generated by running MSD with onlythe backbone conformation from the 4NZU crystal structure to identifyadditional sequences, then feeding these sequences into rigid-bodydocking using RosettaDock (See Gray, J. J. et al. (2003), J. Mol. Biol.;331; 281-299). Low-energy backbone conformations identified byrigid-body docking using the Rosetta Interface Analyzer tool. (SeeLewis, S. M. & Kuhlman, B. A. (2011), PLoS One 6; e20872 and Stranges,P. B. & Kuhlman, B. A (2013), Protein Sci.; 22; 74-82) are then selectedfor the repertoire of alternate conformations. This process is iteratedmultiple times until the difference in measured binding energiesfollowing MSD simulation and rigid-body docking is small. After buildinga repertoire of alternate conformation, several hundred MSD simulationsfollowed by several thousand rigid-body docking runs are performed tocreate a pool of sequences.

Sequences are selected for further analysis by the total energies andbinding energies of the correctly paired interactions and the differencein binding energies between the correctly-paired and mis-pairedinteractions. These sequences are manually examined to identifyfrequently occurring pairs of mutations as well as other mutations thatoften accompany. The identified mutations are divided into multiplefamilies, and within each family, all combinations of possiblealternative mutations are enumerated. For each of these sequences, allfour dimers are then run through Rosetta's “fast relax” protocol (SeeTyka, M. D. et al. (2011), J. Mol. Biol.; 405; 607-618 and Khatib, F. etal. (2011), Proc. Natl. Acad. Sci.; 108; 18949-18953), which allowsbackbone flexibility as it tries to find low-energy conformations. Morethan twenty fast relax trajectories are run for each dimer for eachsequence and the lowest energy conformation for each dimer isidentified. On the basis of the relax simulations, thirty two designpairs (i.e., Aa/Bb pairs) are chosen for an initial round ofexperimental characterization (Table 1).

TABLE 1 Initial Designs for Experimental Characterization^(a) DesignName HC_A (C_(H)1) LC_a (Cκ) HC_B (C_(H)1) LC_b (Cκ) 1.3 S188G S176I — —1.4 S188G S176I S188T — 1.5 S188A S176M — — 1.6 S188A S176M S188T — 1.7S188G S176M — — 1.8 S188G S176M S188T — 1.12 S188G S176I S188I S176G1.13 S188A S176M S188M S176A 1.15 S188I S176A — — 1.18 S188G S176I S188IS176A 2.3 K145A S131R — — 2.4 K145A S131K — — 2.7 K145S S131R — — 2.8K145S S131K — — 3.14 L143A S188M V133F — — 3.15 L143A S188M V133F S188T— 3.18 L143A K145A S131G V133Y — — 3.19 L143A K145A S131G V133Y S188T —5.1 A139L V190G F118I L135F — — 5.2 A139L V190G F118V L135F — — 6.1K145G S188Q S131R — — 6.1 + K145G S188Q S131R S188A S176M 1.5 inv 6.1 +K145G S188Q S131R S188G S176M 1.7 inv 6.2 F122Y K145G S131R — — S188Q6.3 K145G S188Q S131K — — 6.4 F122Y K145G S131K — — S188Q 7.15 F174YV190I L135V T164V — — 7.16 F174Y V190I L135V T164V — T164S S174V 7.17F174Y V190I L135V T164V — T164A S174V 10.1 K145A Q124A S131K F122G L143HQ124Y S131F K145A 10.9 K145A Q124A S131K F122G L143H Q124Y S131Y K145A11.16 F122G L143Y Q124F S131W F122Y S186I T178I K145A V177S ^(a)Allresidues numbered according to Kabat Numbering.

Additionally, through visual inspection of the interface, pairs ofresidues are identified where electrostatic (charge-charge) repulsionmutations could be inserted across the mis-paired interface. Sets ofcharge mutations were made at these positions and run through Rosetta'sfast-relax protocol to ensure that these mutations did not destabilizethe correctly-paired interactions. Following these simulations, a set offour candidate mutations (Table 2) are selected for further experimentalcharacterization.

TABLE 2 Manually-identified, charge-pair mutations aimed atdestabilizing mis-paired interactions^(a) Design HC_A (C_(H)1) LC_a (Cκ)HC_B (C_(H)1) LC_b (Cκ) 12.1 K221E E123K — — 12.2 K221E E123Q — — 13.1 —T129K D146K T129E 13.2 K145E T129K/T180K D146K T129E/T180E ^(a)Allresidues numbered according to Kabat Numbering.

After experimental validation of the initial designs using 2D UPLC (asdescribed in the methods of Example 2, below), combinations of mutationsfrom Design families 1 and 2 (Table 1) are also relaxed to identifydesigns that appear to destabilize both mis-paired interactions. Ninedesigns, (depicted in Table 3) are selected for experimentalcharacterization.

TABLE 3 Combinations of Initial Designs for Characterization^(a) Family1 Family 2 Design Design HC_A (C_(H)1) LC_a (Cκ) HC_B (C_(H)1) LC_b (Cκ)1.3 2.3 K145A S131R — — S188G S176I 1.4 2.3 K145A S131R S188T — S188GS176I 1.5 2.3 K145A S131R — — S188A S176M 1.6 2.3 K145A S131R S188T —S188A S176M 1.7 2.3 K145A S131R — — S188G S176M 1.8 2.3 K145A S131RS188T — S188G S176M 1.12 2.3 K145A S131R S188I S176G S188G S176I 1.182.3 K145A S131R S188I S176A S188G S176I 1.18 2.4 K145A S131K S188I S176AS188G S176I ^(a)All residues numbered according to Kabat Numbering.

Example 2: Synthesis and Characterization of Constructs ContainingC_(H)1/Cκ Designs (Lacking Variable Domains)

A. Cloning of Wild-Type C_(H)1-Fc/Cκ Constructs and Incorporation ofDesigns for Specificity and Stability Screening.

To interrogate the ability of select C_(H)1/Cκ designs (i.e., asdescribed in Example 1) to provide a specific interface thatdiscriminates from wild-type (WT) or alternately designed C_(H)1/Cκinterfaces, it is useful to remove the variable domains to observeC_(H)1/Cκ-specific pairing strengths as previous work in the fieldshowed that the variable domain interface also influences HC/LC pairingspecificity. (Lewis et al., 2014 Nature Biotechnol. 32: 191-198)Additionally, two Cκ constructs are constructed (with or without anN-terminal 8×Histag) to enable charge-based separation of different Cκproteins on a reverse phase HPLC column using the screening methodologydescribed below. The mutations added to the C_(H)1-Fc/Cκ constructs aredesigned to enable improved specific assembly of “Aa” and “Bb” HC/LCpairs, relative to the mis-paired “Ab” and “Ba” HC/LC pairs, where ‘A’is a first C_(H)1-Fc, ‘B’ is a second C_(H)1-Fc, ‘a’ is a first LC Cκconstruct designed to pair more specifically with ‘A’ relative to ‘B’,and ‘b’ is a second LC Cκ construct designed to pair more specificallywith ‘B’ relative to ‘A’ (see Tables 1-3).

Thus, human IgG1 HC and kappa LC constructs lacking variable domaingenes are constructed in pEHG1 and pEHK vectors (Lonza), modifiedin-house for general antibody HC and LC expression. The Wild-Type (nodesigns) pEHG1_C_(H)1-Fc plasmid is generated by direct recombinasecloning of two separate and overlapping GeneBlocks (“gBlocks”,Integrated DNA Technologies (IDT)) coding for the C_(H)1/hinge regionand the C_(H)2-C_(H)3, respectively, into the pEHG1 vector using HindIIIand EcoRI restriction sites. The hinge-encoding region also contains afactor Xa cleavage site to allow isolation of the C_(H)1/Cκ heterodimerfrom the IgG1-Fc region for crystallography applications. (see SEQ IDNO:1) The Wild-Type (no designs) pEHK_Cκ (SEQ ID NO:2) and pEHK_8×HIS_Cκ(SEQ ID NO:3) plasmids are created by recombinase cloning of singlegBlocks (IDT) into the pEHK vector using AgeI and EcoRI restrictionsites.

The computational and rational design modifications are introduced intothe pEHG1_C_(H)1-Fc, pEHK_Cκ, and pEHK_8×HIS_Cκ plasmids in one of twoways. The first procedure employs site directed primer basedmutagenesis. Briefly, the site-directed mutagenesis protocol employs asupercoiled double-stranded DNA vector and two synthetic oligonucleotideprimers (IDT) containing the desired mutation(s). The oligonucleotideprimers, each complementary to opposite strands of the vector, areextended during thermal cycling by DNA polymerase (HotStar HiFidelityKit, Qiagen Cat. #202602) to generate an entirely new mutated plasmid.Following temperature cycling, the product is treated with Dpn I enzyme(New England BioLabs, Cat #R0176). The Dpn I enzyme cleaves onlymethylated parental DNA derived from the parental plasmid which isprepared in E. coli. Each newly generated mutant plasmid pool is thentransformed into E. coli strain TOP 10 competent cells (LifeTechnologies). The second method employs synthesized dual or singlegBlocks (IDT) containing 15 base pair 5′ and 3′ overlaps to allowrecombinase-based cloning into pEHG1_C_(H)1-Fc plasmids usingrestriction sites AgeI and BamHI or cloning into pEHK_Cκ and/orpEHK_8×HIS_Cκ plasmids digested with AgeI and EcoRI. Therecombinase-based cloning is performed using the In-Fusion protocol(Clontech Laboratories, Inc.) to generate the clone. The In-Fusionconstruct is then transformed into E. coli strain TOP 10 competent cells(Life Technologies). Colonies are picked and clonal DNA is produced byminiprepping according to standard procedures (Qiagen MiniPrep), andsequenced in-house. Medium and large scale plasmid purifications areperformed according to the instructions within the Plasmid Plus MidiprepKit (Qiagen Cat. #12965) and Maxiprep Plus Kit (Qiagen Cat. #12965),respectively.

B. Protein Expression in Human Embryonic Kidney Cells (HEK293) orChinese Hamster Ovary (CHO) Cells.

To test designs for their ability to enable specific assembly of selectC_(H)1/Cκ complexes, for each design an ‘A’ or ‘B’ C_(H)1-Fc construct(containing the C_(H)1 design sequence) is co-transfected with both ‘a’and ‘b’ Cκ constructs (containing the Cκ design sequences) for mammaliancell expression and soluble protein secretion. Thus, for each design,the ‘A’ or ‘B’ C_(H)1-Fc HC protein is exposed to both LC Cκ proteins.Expressed protein is purified from the mammalian cell culturesupernatants, reduced, and characterized by gradient reverse phase (RP)HPLC in a two-dimensional chromatography process (described below) todetermine whether the designs induce a preference in pairing of theC_(H)1-Fc proteins with one Cκ protein over the other.

Briefly, constructs are expressed transiently either in HEK293 or CHOcells according to protocols previously described in the literature(Lewis, et al. (2014), Nat. Biotechnol., 32; 191-198 and Rajendra et al.(2015), Biotechnology and Bioengineering, 112; 977-986). To screenselect designs as described in Example 1, for each design, (i)pEHG1_C_(H)1-Fc (containing ‘A’ or ‘B’ side C_(H)1 design sequence),(ii) pEHK Cκ 8×HIS, and (iii) pEHK Cκ plasmids (all lacking variabledomain genes, e.g., “V_(L) Minus”) are all transfected transiently intoHEK293F or CHO cells using a 1:1.5:1.5 plasmid ratio, respectively. The“a” side Cκ design sequences (see Tables 1-3) are typically cloned intothe pEHK Cκ V_(L) Minus plasmid, while the “b” side Cκ design sequencesare typically cloned into the pEHK Cκ 8×HIS V_(L) Minus plasmid.Additionally, for each design screened, each pEHG1_C_(H)1-Fc plasmid(containing either an ‘A’ or ‘B’ side C_(H)1 design sequence) is alsoco-transfected with only its design counterpart pEHK Cκ V_(L) Minus orpEHK Cκ 8×HIS V_(L) Minus plasmid (using a 1:3 HC/LC ratio) as a controlto allow identification of which peak in the reverse phase elutionprofile (described below) belongs to the Cκ protein versus the 8×His_Cκprotein. Transfected cells are grown at 37° C. in a 5% CO₂ incubatorwhile shaking at 125 rpm for 5 days. Secreted protein is harvested bycentrifugation at 2K rpm for 5 min. and recovery of the supernatant.

C. Specificity (Correct Assembly) Screening

The specificity or percent correct assembly of the design pairs may bedetermined using a two-dimension UPLC (2D UPLC)purification/characterization method (tandem protein G+reversephase-high pressure liquid chromatography (HPLC) with in-vial reduction)or by liquid chromatography/mass spectrometry (LCMS).

2D UPLC is performed using a Dionex Ultimate 3000 Dual Rapid SeparationLiquid Chromatography system. Briefly, the first dimension protein Gstep purifies the expressed C_(H)1-Fc/Cκ protein dimer using a protein Gcolumn (POROS® G 20 μm Column, 2.1×30 mm, 0.1 mL part #2-1002-00)equilibrated with 1×PBS prior to sample load. All flow rates are 1mL/min except the final post elution column wash at 2 mL/min. 450 μLsamples of filtered cell culture supernatant is injected onto theprotein G column. After washing with 1×PBS, the column is eluted with100 mM sodium phosphate, pH 2.2 (2 minutes). Protein G eluents arecollected into vials pre-filled with 20 μL 1M TCEP(tris(2-carboxyethyl)phosphine) in an auto sampler held at ambienttemperature. Titers of expressed C_(H)1-Fc/Cκ protein dimer aredetermined by comparison to eluent peak areas obtained from a standardcurve of an in-house human IgG protein G purification.

The second dimension is for characterization of the relative ratio ofeach Cκ protein (“a” side designs in Cκ OR “b” side designs in 8×HIS_Cκ,see Tables 1-3) that binds to each C_(H)1-Fc protein. The methodutilizes two buffers: Buffer A being 100% H₂O and 0.05% trifluoroaceticacid (TFA); Buffer B being 100% acetonitrile (CAN), 0.05% TFA. All flowrates are 1 mL/min. The method injects each purified and reduced sampleonto a Waters Symmetry C18 column, 4.6×75 mm, 3.5 μm (WAT066224)equilibrated in 95% Buffer A/5% Buffer B. Once each sample is capturedonto the column, a gradient is applied starting at 10% ACN and linearlygoing to 40% ACN in 13 minutes. The column is then flushed with up to70% ACN for 2 minutes and re-equilibrated with 5% ACN for 2 minutesprior to the next injection. The reversed-phase profile typicallyincludes three peaks; one for each of the Cκ and Cκ 8×HIS polypeptides,and one for the C_(H)1-Fc polypeptide. Comparisons by reversed-phasechromatography are made by overlaying the control samples (as describedin Step B above) with test samples. The areas under each peak aredetermined to calculate percent correct assembly of the particularC_(H)1/Cκ design pair.

Alternatively, test articles may be analyzed for specific assembly byLCMS. Briefly, test articles may be purified from supernatants using theUPLC method as described above. However, instead of reducing the samplesin TCEP and running the 2^(nd) dimension reverse phase column, thesamples are submitted for LCMS characterization as generally describedpreviously (See Lewis, et al. (2014), Nat. Biotechnol., 32; 191-198).

D. Thermal Stability Determinations

Enzyme-linked immunosorbent assays (ELISAs) or differential-scanningcalorimetry (DSC) assays for the detection of thermo-challenged proteinsamples may be performed to compare the stability of the designedsamples against the Wild-Type control proteins. For ELISAs, briefly,96-well U-bottom high protein binding 96-well plates (Greiner bio-one,cat #650061) are coated overnight at 4° C. with 100 μL/well with 1 μg/mlof Sheep anti human IgG (Fd) (Meridian Life Science Cat. #W90075C-1) ina 0.05 M NaHCO3 buffer, pH 8.3. The plates are then washed four timeswith PBS with 0.1% TWEEN® (PBST) and blocked for 1 hour with casein(Thermo Scientific, cat #37528) at 37° C. The plates are washed againand 100 μL/well of culture supernatants containing the “variable minus”C_(H)1-Fc proteins expressed with their cognate “variable minus” Cκproteins (normalized to be at about 100 ng/ml) are incubated for 1 hr.at 37° C. The supernatants are pre-exposed to various temperatures for 1hr using a Thermal cycler with a 25° C. thermal gradient window (55° C.to 80° C.). The plates are then washed and Goat-anti-human Kappa-HRP(Southern Biotech Cat. #2060-05) at 1:8000 in casein is added andincubated for 1 hr at room temperature. The plates are then washed and 1step Ultra TMB ELISA substrate (Thermo Scientific Cat. #34208) is addedat 50 μL/well. The reaction is allowed to proceed for 1-15 minutes thenquenched by the addition of (50 μL) 2.5M H₂SO₄. The absorbance at 450 nmis then read using a SPECTRAMAX® 190 UV plate reader (MolecularDevices).

Alternatively, the stability of purified proteins (i.e., WT and designconstructs lacking variable domains purified, for example, by FPLC usingProtein A) may be characterized using differential scanning calorimetry(DSC), essentially as follows. The midpoints of the thermal unfoldingtransitions (denoted ‘T_(m)’) of the C_(H)1/C_(κ) domains provide ameasure of their relative stability. DSC is performed using an automatedcapillary DSC system (capDSC, GE Healthcare). Protein solutions andreference (buffer) solutions are sampled automatically from a 96-wellplate using the robotic attachment. Before each protein scan, at leastone buffer/buffer scan is performed to define the baseline forsubtraction. All 96-well plates containing protein are stored within theinstrument at 6° C. Samples are run at 1.0 mg/ml protein concentrationin PBS. Scans are performed from 10 to 110° C. at 90° C./hr using thelow feedback mode. Scans are analyzed using the Origin software suppliedby the manufacturer. Subsequent to the subtraction of reference baselinescans, nonzero protein scan baselines are corrected using a third-orderpolynomial.

E. Specificity of Assembly and Thermal Stability CharacterizationResults

Select initial and combination designs in the Cκ/C_(H)1-Fc constructslacking variable domains are evaluated for specificity of correctassembly as determined by reverse phase HPLC and/or LCMS according toprocedures as described above. Results of specific assemblycharacterizations are provided in the Table 4 below.

TABLE 4 HC_A LC_a HC_B LC_b Design C_(H)1 Cκ C_(H)1 Cκ % Aa % Ab % Ba %Bb Initial designs evaluated using reverse phase HPLC^(a) WT WT WT WT WT76.0 ± 6.6  23.9 ± 6.7  76.6 ± 2.7  23.3 ± 2.7 1.3 S188G S176I WT WT100/95    0/2.4 91.6/73   8.4/25  1.4 S188G S176I S188T WT 100   0  82.2 17.8 1.5 S188A S176M WI WT 96.3 3.7 92.1 7.9 1.6 S188A S176M S188TWT 94.4/87.1 5.6/7.4 78.3/61.7 21.7/27.2 1.7 S188G S176M WT WT 100/1000/0 91.9/94   8.1 1.8 S188G S176M S188T WT  100/96.7 0/0 77.7/94.2 23.31.12 S188G S176I S188I S176G  94 ± 1.5 3.8 ± 3.6 67.8 ± 6.3  30.7 ± 4.11.13 S188A S176M S188M S176A 89.4 10.6  67.8 32.2 1.15 S188I S176A WT WT72.2/68.4 27.8   0/100 0/0 1.18 S188G S176I S188I S176A 98.7 ± 2.3  0 ±0 68.2 ± 3.1  27.9 ± 5.2 2.3 K145A S131R WT WT  59 ± 5.9  41 ± 5.9 1.3 ±2.2 96.3 ± 3.7 2.4 K145A S131K WT WT 61.6 38.4  0 100 2.7 K145S S131R WTWT 51.8 48.2  0 100 2.8 K145S S131K WT WT 34.8 65.2  7.5 92.5 12.1 K221EE123K WT WT 100   0   23.8^(b) 76.2^(b) 12.2 K221E E123Q WT WT 69.130.9  68 32 Combination designs evaluated using reverse phase HPLC^(a)1.3.1 K145A S131R WT WT 77.7 22.3  12.5 87.5 S188A S176I 1.12.1 K145AS131R S188I S176G 72.3 27.7  36.3 63.7 S188A S176I 1.18.1 K145A S131RS188I S176A 70.1 29.9  40 60 S188A S176I 14.1.2 K145A S131R WT WT100^(b ) 0^(b)  4.5^(b) 95.5^(b) K221E E123K 14.3.1.1 S188A S176I K221EE123K 94.7 5.3 0 100 K145A S131R 14.3.1.2 S188G S176I K221E E123K 100  0   0 100 K145A S131R 15.1 K145A S131R WT WT 97.3 2.7 5.8 94.2 K221EE123K S188A S176I 15.2 K145A S131R WT WT 95.7 4.3 6.1 93.9 K221E E123KS188G S176I Combination designs evaluated by LCMS^(c) 14.1.2 K145A S131RWT WT 99.8 ± 0.1   0.2 ± 0.04 0/0 100/100 K221E E123K 14.3.1.1 S188AS176I K221E E123K 98.2 ± 0.6  1.8 ± 0.6 0.2 ± 0.2 99.8 ± 0.2 K145A S131R14.3.1.2 S188G S176I K221E E123K 99.8 0.2 0.1 99.0 K145A S131R^(a)Values are calculated based on area for each Cκ species, ‘a’ or ‘b’,as observed using denaturing, reverse phase chromatography with proteinG purified samples that are reduced prior to injection onto the reversephase column. Values with error (±) were run 3 or more times. The meanvalue is listed followed by the standard deviation. Cells with 2 valueswere run in duplicate and the values listed are from each replicate.^(b)Peak overlap in the HPLC method made these values difficult toquantify. ^(c)Values are calculated based on the deconvoluted peak areasfor each of the species from the LCMS evaluation. Values with error (±)were run 3 or more times. The mean value is listed followed by thestandard deviation. Cells with 2 values were run in duplicate and thevalues listed are from each replicate.

The Wild-Type (WT) ‘b’ Cκ with the 8×Histag expresses slightly poorerthan the WT ‘a’ Cκ without the tag resulting in a roughly 75/25 ratio inthe absence of any designs (Table 4). Many of the initial designs showmodest increases in “Aa” and/or “Bb” assembly but are not consideredfurther due to decreases in expression, likely due to destabilization ofthe interface. However, certain designs express well and improve thepercent of “Aa” and/or “Bb” assembly. These include designs 1.3, 1.18,2.3, and 12.1. A second round of designs combining the best mutationsfrom the initial screen are generated in the same Cκ/C_(H)1-Fcconstructs. Combining certain designs results in modest increases inspecificity of assembly over the initial designs. Many combinationdesigns again result in reduced protein expression and are notconsidered further. Certain designs, however, result in near completespecificity of pairing with ‘A’ C_(H)1-Fc assembling with near completespecificity to ‘a’ Cκ, and ‘B’ C_(H)1-Fc assembling with near completespecificity to ‘b’ Cκ. These designs include 14.1.2, 14.3.1.1, 14.3.1.2,15.1, and 15.2 (Table 4). To provide a secondary method for quantitatingthe assembly of the Cκ/C_(H)1-Fc proteins, a subset of the designs(14.1.2, 14.3.1.1, and 14.3.1.2) are submitted for LCMS analyses. Basedon the mass spectrometry results, each of these designs show nearcomplete specificity of assembly of “Aa” and “Bb” relative to themis-paired “Ab” and “Ba” species, similar to the data produced using thereverse phase HPLC method (Table 4).

Species of select combination designs in the Cκ/C_(H)1-Fc constructslacking variable domains (i.e. “Aa” or “Bb” dimers) are evaluated forthermal stability using DSC according to procedures essentially asdescribed above. Results of thermal stability characterizations areprovided in the Table 5 below.

TABLE 5 C_(H)1-Fc/Cκ Midpoint of Heterodimer thermal denaturation (WT orDesign) (T_(m)) in ° C. WT 71.9 14.1.2Aa 70.3 14.3.1.1Aa 78.5 14.3.1.1Bb71.1 15.1Aa 78.8 15.2Aa 77.4

The 14.1.2, 14.3.1.1, 15.1, and 15.2 design constructs do not result inreduced stability compared to the wild-type C_(H)1/Cκ domains (Table 5).C_(H)1/Cκ domains containing either C_(H)1_S188A/Cκ_S176I orC_(H)1_S188I/Cκ_S176A are found to be stabilized over WT C_(H)1/Cκ basedon their midpoints of thermal denaturation (T_(m)) measured using DSC.14.3.1.1 and 15.1 both contain C_(H)1_S188A/Cκ_S176I and aresignificantly more stable than the WT C_(H)1/Cκ heterodimer (Table 5).In summary, the data in Table 5 demonstrates that the indicated designs,which each enable specific C_(H)1/Cκ assembly, are essentially asstable, or more stable than the WT heterodimer.

Example 3: Bispecific Antibodies

A. Cloning of IgG BsAbs Harboring Novel C_(H)1/Cκ Designs

Human IgG1 bispecific antibodies containing select C_(H)1/Cκ designmutations or WT C_(H)1/Cκ are constructed using pEHG1 (HC) and pEHK (LC)vectors, as generally described above. Variable domains from parentalmAbs Pertuzumab (Franklin, M. C. et al. (2004), Cancer Cell; 5;317-328), MetMAb (Merchant, M., et al. (2013), Proc. Nat'l. Acad. Sci.USA; 110; E2987-E2996), Matuzumab (Schmiedel, J., et al. (2008), CancerCell; 13; 365-373) and BHA10 (Jordan, J. L., et al. (2009), Proteins;77; 832-841) are used in preparation of test articles (fully IgGbispecific antibodies). The Pertuzumab and MetMAb parental mAbconstructs are chosen as the receptacles for the HC_“A” and LC_“a”design sequences for each of the C_(H)1/Cκ designs tested, while theBHA10 and Matuzumab parental mAb constructs are chosen as thereceptacles for the HC_“B” and LC_“b” design sequence for each of theC_(H)1/Cκ designs tested. Design mutations are introduced using eitherthe site-directed mutagenesis or gBlock recombinase cloning methods,also as described above. Each bispecific antibody is further engineeredto contain select C_(H)3 domain design mutations (e.g., Design 7.8.60 asdescribed in Leaver-Fay A., et al. (2016), Structure; 24; 641-651 and WO2016/118742 (A1)) to improve HC heterodimerization, as well as an N297Qmutation in each HC to reduce N-linked glycosylation. Additionally,select variable domain design mutations (e.g. Designs “AB” or “H4DR”,each as described in WO2014/150973) are each separately and individuallyintroduced into one of the V_(H) V_(L) interfaces of each bispecificantibody to evaluate the impact of specificity designs in both thevariable and constant domains of the Fabs. Plasmid isolation, sequencingand scale-up are essentially as described above for constructs lackingvariable domains

B. Protein Expression

For IgG bispecific antibody production, four plasmids (two that containone of the two HC encoding sequences (i.e., HC_“A” or HC_“B”) and twothat contain one of the two LC encoding sequences (i.e., LC_“a” andLC_“b”)) are co-transfected in HEK293F cells (using 1:3 HC:LC plasmidratios) or CHO cells (using 1:1 HC:LC plasmid ratios). Transfected cellsare grown at 37° C. in an 8% CO₂ incubator while shaking at 125 rpm for5 days (HEK293F cells) or 6 days (CHO cells). For both HEK293 and CHO,secreted protein material is harvested by centrifugation at 5 K rpm for5 min at the end of the culture period. Supernatants are passed through0.22 m filters (small scale) for either large or small scalepurification.

Table 6 below provides the parental mAb components and SequenceIdentification Numbers (SEQ ID NOs.) of the complete HCs and LCs of thefully IgG bispecific antibodies constructed with, and without, selectC_(H)1/Cκ designs as described herein.

TABLE 6 C_(H)1/Cκ HC_A LC_a HC_B LC_b design (SEQ ID NO.) (SEQ ID NO.)(SEQ ID NO.) (SEQ ID NO.) MetMAb^(a) × BHA10^(b) Wild-Type 4 10 28 3014.1.2 5 11 28 30 14.3.1.1 6 12 29 31 14.3.1.2 7 12 29 31 15.1 8 13 2830 15.2 9 13 28 30 Pertuzumab^(a) × BHA10^(b) Wild-Type 14 20 28 3014.1.2 15 21 28 30 14.3.1.1 16 22 29 31 14.3.1.2 17 22 29 31 15.1 18 2328 30 15.2 19 23 28 30 MetMAb^(a) × Matuzumab^(b) Wild-Type 4 10 24 2614.1.2 5 11 24 26 14.3.1.1 6 12 25 27 14.3.1.2 7 12 25 27 15.1 8 13 2426 15.2 9 13 24 26 Pertuzumab^(a) × Matuzumab^(b) Wild-Type 14 20 24 2614.1.2 15 21 24 26 14.3.1.1 16 22 25 27 14.3.1.2 17 22 25 27 15.1 18 2324 26 15.2 19 23 24 26 ^(a)MetMAb and Pertuzumab HCs and LCs contain theHC_“A” and LC_“a” design sequences for each of the C_(H)1/Cκ designs,the AB variable domain designs described previously (WO2014/150973 A1)and 7.8.60A C_(H)3 domain heterodimerization designs also describedpreviously (WO 2016/118742 (A1)). ^(b)BHA10 and Matuzumab HCs and LCscontain the HC_“B” and LC_“b” design sequence for each of the C_(H)1/Cκdesigns, the H4DR variable domain designs described previously(WO2014/150973 A1) and 7.8.60B C_(H)3 domain heterodimerization designsalso described previously (WO 2016/118742 (A1)).

C. Specificity (Correct Assembly) Screening of Fully IgG BispecificAntibodies

The IgG BsAb HC and LC constructs are generated and expressed asdescribed above. Secreted supernatants are purified using the HPLCprotein G method as described above for purifying the Cκ/C_(H)1-Fcproteins, however, the purified proteins are not reduced and submittedfor reverse phase chromatography but rather are collected and submittedfor LCMS analysis essentially as described in Lewis et al., 2014 NatureBiotechnol; 32; 191-198. Table 7 below provides the percent assembly ofthe correctly paired BsAb as well and the mis-paired species containingtwo LC_“a” or two LC_“b” light chains as well as those containingHC_“A”/HC_“A” and HC_“B”/HC_“B” homodimers. All values in the tablerepresent the mean and standard deviation of at least three separateexperiments.

TABLE 7 % % C_(H)1/Cκ % BsAb % 2× LC_a % 2× LC_b HC_A/HC_A HC_B/HC_Bdesign (correct) (mis-pair) (mis-pair) (homodimer) (homodimer)MetMAb^(a) × BHA10^(b) Wild-Type 91.9 ± 3.8 0.8 ± 2.0 4.7 ± 4.7 1.2 ±1.3 1.5 ± 2.8 14.1.2 95.4 ± 0.4 0.0 ± 0.0 3.9 ± 0.3 0.7 ± 0.7 0.0 ± 0.014.3.1.1 93.2 ± 1.6 0.0 ± 0.0 4.6 ± 0.9 2.5 ± 0.9 0.0 ± 0.0 14.3.1.285.6 ± 4.6 0.0 ± 0.0 7.2 ± 2.3 8.4 ± 3.1 0.0 ± 0.0 15.1 87.4 ± 2.5 2.3 ±4.0 8.3 ± 2.1 0.0 ± 0.0 1.9 ± 1.7 15.2 91.4 ± 0.5 0.0 ± 0.0 7.2 ± 0.51.6 ± 0.1 0.0 ± 0.0 Pertuzumab^(a) × BHA10^(b) Wild-Type 58.9 ± 2.8 39.9± 2.7  0.4 ± 0.4 0.0 ± 0.0 0.8 ± 0.9 14.1.2 95.9 ± 2.0 0.0 ± 0.0 0.0 ±0.0 0.0 ± 0.0 4.1 ± 2.0 14.3.1.1 88.3 ± 7.5 1.0 ± 0.1 0.0 ± 0.0 9.2 ±8.0 1.7 ± 0.3 14.3.1.2 84.0 ± 1.7 0.9 ± 0.3 0.0 ± 0.0 14.1 ± 0.8  1.3 ±1.2 15.1 96.8 ± 5.5 0.0 ± 0.0 0.8 ± 1.4 0.8 ± 1.4 1.6 ± 2.8 15.2 94.6 ±3.7 0.0 ± 0.0 2.1 ± 0.9 1.7 ± 2.9 1.8 ± 3.1 MetMAb^(a) × Matuzumab^(b)Wild-Type 91.0 ± 2.6 3.2 ± 2.3 1.2 ± 1.3 1.1 ± 1.2 3.7 ± 2.5 14.1.2 94.8± 0.3 0.0 ± 0.0 1.4 ± 0.3 2.3 ± 0.3 1.7 ± 0.1 14.3.1.1 93.7 ± 0.3 0.8 ±0.1 1.4 ± 0.1 3.4 ± 0.1 0.6 ± 0.3 14.3.1.2 92.6 ± 0.5 0.9 ± 0.2 1.3 ±0.2 5.4 ± 0.3 0.0 ± 0.0 15.1 92.3 ± 2.2 0.0 ± 0.0 4.3 ± 1.3 3.1 ± 1.90.6 ± 0.7 15.2 96.3 ± 2.6 0.0 ± 0.0 1.2 ± 0.6 0.3 ± 0.5 2.2 ± 2.5Pertuzumab^(a) × Matuzumab^(b) Wild-Type  70.0 ± 11.0 21.6 ± 8.0  0.0 ±0.0 0.0 ± 0.0 8.5 ± 4.0 14.1.2 97.3 ± 2.0 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.22.6 ± 1.9 14.3.1.1 96.3 ± 1.5 0.0 ± 0.0 0.0 ± 0.0 2.6 ± 0.6 1.1 ± 1.014.3.1.2 96.2 ± 0.9 0.0 ± 0.0 0.2 ± 0.3 3.7 ± 1.2 0.0 ± 0.0 15.1 99.5 ±0.9 0.0 ± 0.0 0.0 ± 0.0 0.5 ± 0.9 0.0 ± 0.0 15.2 99.1 ± 0.1 0.0 ± 0.00.0 ± 0.0 1.0 ± 0.1 0.0 ± 0.0 ^(a)MetMAb and Pertuzumab HCs and LCscontain the HC_“A” and LC_“a” design sequences for each of the C_(H)1/Cκdesigns, the AB variable domain designs described previously(WO2014/150973 A1) and 7.8.60A C_(H)3 domain heterodimerization designsalso described previously (WO 2016/118742 (A1)). ^(b)BHA10 and MatuzumabHCs and LCs contain the HC_“B” and LC_“b” design sequence for each ofthe C_(H)1/Cκ designs, the H4DR variable domain designs describedpreviously (WO2014/150973 A1) and 7.8.60B C_(H)3 domainheterodimerization designs also described previously (WO 2016/118742(A1)).

The data in Table 7 indicates that all of the indicated designs resultedin >80% correctly paired BsAb assembly for each of the constructsprepared. Further, for the Pertuzumab component-containing constructs,all of the tested C_(H)1/Cκ designs displayed large and significantincreases in percent correct assembly of BsAb compared to their WTC_(H)1/Cκ domain counterparts. (Table 7).

D. Bispecific Antibody Binding Activity Assay

The dual-binding activity of the same synthesized IgG BsAbs is assessedusing a sandwich ELISA. Two Sandwich ELISAs are developed for detectingthe four different BsAb test articles; one for detectinganti-HER-2/anti-EGFR BsAb activity, one for detectinganti-HER-2/anti-LTPR BsAb activity, one for detectinganti-cMET/anti-EGFR BsAb activity and one for detectinganti-cMET/anti-LTPR BsAb activity. For all ELISAs, clear 96-well roundbottom high binding Immulon microtiter plates (Greiner bio-one, cat#650061) are coated overnight at 2-8° C. with 50 μL/well 1 μg/mLhHER-2-Fc or 1 μg/mL hHGFR(cMet)-Fc (both from R&D systems) in a 50 mMNa2CO3 pH 8.3 buffer. The plates are washed 4 times with PBST andblocked with 200 μL/well casein buffer (Pierce) for 1 hr at roomtemperature. The plates are then washed 4 times with PBST and theparental IgG controls or BsAb IgG test articles are added at 50 μL/welland μg/mL and serially diluted 1:3 down the plate. The controls and testarticles are incubated on the plate for 1 hr at room temperature. Theplates are then washed 4 times with PBST and 50 μL/well 1 μg/mLhEGFR-Fc-biotin or hLTβR-Fc-biotin (both from R&D systems) is added for1 hr at room temperature. The plates are then washed 4 times with PBSTfollowed by the addition of a 50 μL/well streptavidin-AP (JacksonImmunoresearch Labs Cat. #016-050-084) diluted 1:1000 in casein buffer.The streptavidin-AP is incubated in each well for 1 hr at roomtemperature. The plates are then washed 4 times with PBST and 100μL/well 1-step PNPP substrate is added (Thermo Scientific Cat. #37621).After approximately 5-15 minutes, plates are read for Absorbance (405nm) using a SPECTRAMAX® UV plate reader (Molecular Devices).Biotin-labeling of the hEGFR-Fc and hLTβR-Fc proteins is performed usingEZ-LINK™ Sulfo-NHS-LC-Biotin (Thermo Scientific Cat. #21327) accordingto the manufacturer's protocol. The BsAb titrations are fit to yieldEC₅₀ values that are listed in Table 8 below.

TABLE 8 Dual Antigen Binding C_(H)1/Cκ design EC50 (μg/mL) MetMAb^(a) ×BHA10^(b) Wild-Type 0.05 14.1.2 0.05 14.3.1.1 0.070 14.3.1.2 0.11 15.1n.d. 15.2 n.d. Pertuzumab^(a) × BHA10^(b) Wild-Type 0.06 14.1.2 0.0314.3.1.1 0.04 14.3.1.2 0.04 15.1 n.d. 15.2 n.d. MetMAb^(a) ×Matuzumab^(b) Wild-Type 0.12 14.1.2 0.10 14.3.1.1 0.20 14.3.1.2 0.2515.1 n.d. 15.2 n.d. Pertuzumab^(a) × Matuzumab^(b) Wild-Type 0.17 14.1.20.19 14.3.1.1 0.15 14.3.1.2 0.10 15.1 n.d. 15.2 n.d. ^(a)MetMAb andPertuzumab HCs and LCs contain the HC_“A” and LC_“a” design sequencesfor each of the C_(H)1/Cκ designs, the AB variable domain designsdescribed previously (WO2014/150973 A1) and 7.8.60A C_(H)3 domainheterodimerization designs also described previously (WO 2016/118742(A1)). ^(b)BHA10 and Matuzumab HCs and LCs contain the HC_“B” and LC_“b”design sequence for each of the C_(H)1/Cκ designs, the H4DR variabledomain designs described previously (WO2014/150973 A1) and 7.8.60BC_(H)3 domain heterodimerization designs also described previously (WO2016/118742 (A1)).

All of the IgG BsAbs proteins showed strong bispecific binding activitytowards their target antigens, but not towards the mis-matched antigensandwich pairs. Some of the EC₅₀s for the BsAbs are lower than what isobserved for others (Table 8). This seems to track closely with thelevel of half-antibody (non-covalently bound single HC/LC species)generation that occurs when one HC/LC pair expresses at a higher levelthan the other HC/LC pair in solution.

E. Fully IgG Bispecific Antibodies without Variable Domain Designs.

To assess the impact of the C_(H)1/Cκ designs to improve IgG BsAbassembly in the absence of variable domain designs (e.g., Design “AB”and “H4DR” as described in WO2014/150973 A1), fully IgG BsAbs areconstructed incorporating a subset of the C_(H)1/Cκ designs describedherein (i.e., Designs 14.1.2, 14.3.1.1, and 14.3.1.2). In addition tothe C_(H)1/Cκ designs, each of the constructs also contains C_(H)3domain design mutations to improve HC heterodimerization (i.e., Design7.8.60 as described in Leaver-Fay et al (2016) and WO 2016/118742 A1),as well as an N297Q mutation in each HC to reduce N-linkedglycosylation. Cloning, expression, and purification of the testarticles is performed essentially as described above. Also as describedabove, the Pertuzumab and MetMAb parental mAb constructs are chosen asthe receptacles for the HC_“A” and LC_“a” design sequences for each ofthe C_(H)1/Cκ designs tested, while the BHA10 and Matuzumab parental mAbconstructs are chosen as the receptacles for the HC_“B” and LC_“b”design sequence for each of the C_(H)1/Cκ designs tested.

Table 9 below provides the parental mAb components and SequenceIdentification Numbers (SEQ ID NOs.) of the complete HCs and LCs of thefully IgG bispecific antibodies constructed with, and without, selectC_(H)1/Cκ designs as described herein (but without any variable domaindesigns).

TABLE 9 HC_A LC_a HC_B LC_b C_(H)1/Cκ design (SEQ ID NO.) (SEQ ID NO.)(SEQ ID NO.) (SEQ ID NO.) MetMAb × BHA10 Wild-Type 32 36 50 52 14.1.2 3337 50 52 14.3.1.1 34 38 51 53 14.3.1.2 35 38 51 53 Pertuzumab × BHA10Wild-Type 39 43 50 52 14.1.2 40 44 50 52 14.3.1.1 41 45 51 53 14.3.1.242 45 51 53 MetMAb × Matuzumab Wild-Type 32 36 46 48 14.1.2 33 37 46 4814.3.1.1 34 38 47 49 14.3.1.2 35 38 47 49 Pertuzumab × MatuzumabWild-Type 39 43 46 48 14.1.2 40 44 46 48 14.3.1.1 41 45 47 49 14.3.1.242 45 47 49

The fully IgG BsAbs containing the C_(H)1/Cκ Designs 14.1.2, 14.3.1.1,and 14.3.1.2 and the C_(H)3 Design 7.8.60 (but lacking variable domaindesigns) are similarly characterized for specificity of assembly usingLCMS as described above and in Lewis et al., 2014 Nature Biotechnol. 32:191-198. Table 10 below provides the percent assembly of the correctlypaired BsAb as well and the mis-paired species containing two LC_“a” ortwo LC_“b” light chains as well as those containing HC_“A”/HC_“A” andHC_“B”/HC_“B” homodimers. All values in the table represent the mean andstandard deviation of at least three separate experiments.

TABLE 10 % C_(H)1/Cκ % BsAb % 2× LC_a % 2× LC_b HC_A/HC_A % HC_B/HC_Bdesign (correct) (mis-pair) (mis-pair) (homodimer) (homodimer)MetMAb^(a) × BHA10^(b) Wild-Type 61.0 ± 1.0 14.6 ± 1.2 24.4 ± 0.4  0.0 ±0.0 0.0 ± 0.0 14.1.2 71.9 ± 2.1  2.7 ± 0.5 25.4 ± 1.8  0.0 ± 0.0 0.0 ±0.0 14.3.1.1 65.5 ± 1.2  2.7 ± 0.1 31.8 ± 1.2  0.0 ± 0.0 0.0 ± 0.014.3.1.2 68.5 ± 2.3  2.8 ± 1.0 28.8 ± 1.5  0.0 ± 0.0 0.0 ± 0.0Pertuzumab^(a) × BHA10^(b) Wild-Type 59.0 ± 5.3 33.9 ± 7.8 7.1 ± 2.6 0.0± 0.0 0.0 ± 0.0 14.1.2 57.5 ± 1.7 36.0 ± 1.8 6.5 ± 0.4 0.0 ± 0.0 0.0 ±0.0 14.3.1.1 68.2 ± 0.3 28.3 ± 0.2 3.6 ± 0.2 0.0 ± 0.0 0.0 ± 0.014.3.1.2 71.3 ± 2.6 26.9 ± 1.3 1.8 ± 1.5 0.0 ± 0.0 0.0 ± 0.0 MetMAb^(a)× Matuzumab^(b) Wild-Type 78.6 ± 0.5 12.3 ± 0.7 9.0 ± 0.9 0.0 ± 0.0 0.3± 0.3 14.1.2 92.3 ± 0.4  1.5 ± 0.4 5.6 ± 0.2 0.0 ± 0.0 0.5 ± 0.214.3.1.1 80.7 ± 2.2  0.0 ± 0.0 19.1 ± 2.3  0.0 ± 0.0 0.0 ± 0.0 14.3.1.2n.d.^(c) n.d.^(c) n.d.^(c) n.d.^(c) n.d.^(c) Pertuzumab^(a) ×Matuzumab^(b) Wild-Type 64.1 ± 5.1 29.1 ± 7.2 5.1 ± 1.8 2.9 ± 0.5 0.0 ±0.0 14.1.2 81.3 ± 0.8 18.1 ± 1.7 0.0 ± 0.0 0.7 ± 1.2 0.0 ± 0.0 14.3.1.180.0 ± 2.0 11.5 ± 0.5 8.5 ± 2.3 0.0 ± 0.0 0.0 ± 0.0 14.3.1.2 78.6 ± 1.012.4 ± 0.2 9.0 ± 1.2 0.0 ± 0.0 0.0 ± 0.0 ^(a)MetMAb and Pertuzumab HCsand LCs contain the HC_“A” and LC_“a” design sequences for each of theC_(H)1/Cκ designs, and 7.8.60A C_(H)3 domain heterodimerization designsalso described previously (WO 2016/118742 (A1)). ^(b)BHA10 and MatuzumabHCs and LCs contain the HC_“B” and LC_“b” design sequence for each ofthe C_(H)1/Cκ designs and 7.8.60B C_(H)3 domain heterodimerizationdesigns also described previously (WO 2016/118742 (A1)). n.d. = notdone.

The data in Table 10 indicates that the C_(H)1/Cκ domains generallyimprove correct HC/LC pairing within fully IgG BsAb even in the absenceof variable domain designs in the Fab regions. In the majority of cases,incorporating the C_(H)1/Cκ designs alone in the Fab improves correctHC/LC pairing, though some increases are small and the percent correctassembly levels for the full IgG BsAb do not generally achieve thelevels observed when the variable domain designs are also included inthe Fab regions.

Sequences

pEHG1_C_(H)1-Fc SEQ ID. NO. 1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDK GSIEGRGSTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG. pEHK_Cκ SEQ ID. NO. 2RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC.pEHK_8XHIS_Cκ SEQ ID. NO. 3HHHHHHHHGGGGSTGRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. MetMAb VH AB, CH1 WT, N297Q, 7.8.60ASEQ. ID. NO. 4 EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVR K APGKGLEWVGMIDPSNSDTRFNP E FKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMT DNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL M SDGSFFL ASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.MetMAb VH AB, CH1 14.1.2 (K145A K221E), N297Q, 7.8.60A SEQ. ID. NO. 5EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVR K APGKGLEWVGM IDPSNSDTRFNP EFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV A DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVD EKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMT DNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL M SDGSFFL ASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.MetMAb VH AB, CH1 14.3.1.1A (K145A S188A), N297Q, 7.8.60A SEQ. ID. NO. 6EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVR K APGKGLEWVGM IDPSNSDTRFNP EFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV A DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL A SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMT DNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL M SDGSFFL ASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.MetMAb VH AB, CH1 14.3.1.2A (K145A S188G), N297Q, 7.8.60A SEQ. ID. NO. 7EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVR K APGKGLEWVGM IDPSNSDTRFNP EFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV A DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL G SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMT DNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL M SDGSFFL ASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.MetMAb VH AB, CH1 15.1 (K145A S188A K221E), N297Q, 7.8.60ASEQ. ID. NO. 8 EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVR K APGKGLEWVGMIDPSNSDTRFNP E FKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV A DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL A SVVTVPSSSLGTQTY ICNVNHKPSNTKVD EKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMT DNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL M SDGSFFL ASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.MetMAb VH AB, CH1 15.2 (K145A S188G K221E), N297Q, 7.8.60ASEQ. ID. NO. 9 EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVR K APGKGLEWVGMIDPSNSDTRFNP E FKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV A DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL G SVVTVPSSSLGTQTY ICNVNHKPSNTKVD EKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMT DNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL M SDGSFFL ASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG. MetMAb VL AB, CK WTSEQ. ID. NO. 10 R IQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQ D KPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC.MetMAb VL AB, CK 14.1.2 (E123K S131R) SEQ. ID. NO. 11 RIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQ D KPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTVAAPSVFIFPPSD K QLKSGTA R VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC.MetMAb VL AB, CK 14.3.1.1A and 14.3.1.2A (S131R S176I) SEQ. ID. NO. 12 RIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQ D KPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA R VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL I STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. MetMAb VL AB, CK 15.1 and 15.2 (E123K S131R S176I)SEQ. ID. NO. 13 R IQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQ D KPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTVAAPSVFIFPPSD K QLKSGTA R VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL I STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. Pertuzumab VH AB, CH1 WT, N297Q, 7.8.60ASEQ. ID. NO. 14 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR K APGKGLEWVADVNPNSGGSIYNQ E FKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMT DNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL M SDGSFFL ASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.Pertuzumab VH AB, CH1 14.1.2 (K145A K221E), N297Q, 7.8.60ASEQ. ID. NO. 15 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR K APGKGLEWVADVNPNSGGSIYNQ E FKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV A DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVD EKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMT DNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL M SDGSFFL ASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.Pertuzumab VH AB, CH1 14.3.1.1A (K145A S188A), N297Q, 7.8.60ASEQ. ID. NO. 16 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR K APGKGLEWVADVNPNSGGSIYNQ E FKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV A DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL A SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMT DNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL M SDGSFFL ASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.Pertuzumab VH AB, CH1 14.3.1.2A (K145A S188G), N297Q, 7.8.60ASEQ. ID. NO. 17 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR K APGKGLEWVADVNPNSGGSIYNQ E FKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV A DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL G SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMT DNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL M SDGSFFL ASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.Pertuzumab VH AB, CH1 15.1 (K145A S188A K221E), N297Q, 7.8.60ASEQ. ID. NO. 18 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR K APGKGLEWVADVNPNSGGSIYNQ E FKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV A DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL A SVVTVPSSSLGTQTY ICNVNHKPSNTKVD EKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMT DNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL M SDGSFFL ASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.Pertutumab VH AB, CH1 15.2 (K145A S188G K221E), N297Q, 7.8.60ASEQ. ID. NO. 19 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR K APGKGLEWVADVNPNSGGSIYNQ E FKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV A DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL G SVVTVPSSSLGTQTY ICNVNHKPSNTKVD EKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMT DNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL M SDGSFFL ASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG. Pertuzumab VL AB, CK WTSEQ. ID. NO. 20 R IQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQ D KPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.Pertuzumab VL AB, CK 14.1.2 (E123K S131R) SEQ. ID. NO. 21 RIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQ D KPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSD K QLKSGTA R VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.Pertuzumab VL AB, CK 14.3.1.1A and 14.3.1.2A (S131R S176I)SEQ. ID. NO. 22 R IQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQ D KPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA R VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL I STLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.Pertuzumab VL AB, CK 15.1 and 15.2 (E123K S131R S176I) SEQ. ID. NO. 23 RIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQ D KPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSD K QLKSGTA R VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL I STLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.Matuzumab VH H4DR, CH1 WT, N297Q, 7.8.60B SEQ. ID. NO. 24QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVR Y APGQGLEWIGEFNPSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRD YDYDGRYFDYWG RGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR R P R VYTLPPSREEMTKNQVSLV CLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYS VLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G.Matuzumab VH H4DR, CH1 14.3.1.1B and 14.3.1.2B (K221E), N297Q, 7.8.60BSEQ. ID. NO. 25 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVR Y APGQGLEWIGEFNPSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRD YDYDGRYFDYWG RGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVD EKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR R P R VYTLPPSREEMTKNQVSLV CLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYS VLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G. Matuzumab VL H4DR, CK WTSEQ. ID. NO. 26 DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQ R KPG D APKLLIYDTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC.Matuzumab VL H4DR, CK 14.3.1.1B and 14.3.1.2B (E123K) SEQ. ID. NO. 27DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQ R KPG D APKLLIYDTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTFGQG TKVEIKRTVAAPSVFIFPPSDK QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC.BHA10 VH H4DR, CH1 WT, N297Q, 7.8.60B SEQ. ID. NO. 28QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVR Y APGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSW EGFPYWG RGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR R P R VYTL PPSREEMTKNQVSL VCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYS VLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG. BHA10 VH H4DR, CH1 14.3.1.1B and14.3.1.2B (K221E), N297Q, 7.8.60B SEQ. ID. NO. 29QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVR Y APGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSW EGFPYWG RGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVD EKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR R P R VYTL PPSREEMTKNQVSL VCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYS VLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG. BHA10 VL H4DR, CK WTSEQ. ID. NO. 30 DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQ R KPG D APKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.BHA10 VL H4DR, CK 14.3.1.1B and 14.3.1.2B (E123K) SEQ. ID. NO. 31DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQ R KPG D APKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKRTVAAPSVFIFPPSD K QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.MetMAb VH WT, CH1 WT, N297Q, 7.8.60A SEQ. ID. NO. 32EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMT DNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL M SDGSFFL ASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.MetMAb VH WT, CH1 14.1.2 (K145A K221E), N297Q, 7.8.60A SEQ. ID. NO. 33EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV A DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVD EKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMT DNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL M SDGSFFL ASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.MetMAb VH WT, CH1 14.3.1.1A (K145A S188A), N297Q, 7.8.60ASEQ. ID. NO. 34 EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV A DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL A SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMT DNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL M SDGSFFL ASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.MetMAb VH WT, CH1 14.3.1.2A (K145A S188G), N297Q, 7.8.60ASEQ. ID. NO. 35 EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV A DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL G SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMT DNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL M SDGSFFL ASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG. MetMAb VL WT, CK WTSEQ. ID. NO. 36 DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC.MetMAb VL WT, CK 14.1.2 (E123K S131R) SEQ. ID. NO. 37DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTVAAPSVFIFPPSD K QLKSGTA R VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC.MetMAb VL WT, CK 14.3.1.1A and 14.3.1.2B (S131R S176I) SEQ. ID. NO. 38DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA R VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL I STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. Pertuzumab VH, CH1 WT, N297Q, 7.8.60ASEQ. ID. NO. 39 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMT DNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL M SDGSFFL ASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.Pertuzumab VH WT, CH1 14.1.2 (K145A K221E), N297Q, 7.8.60ASEQ. ID. NO. 40 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV A DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVD EKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMT DNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL M SDGSFFL ASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG. Pertuzumab VH WT, CH1 14.3.1.1A(K145A S188A), N297Q, 7.8.60A SEQ. ID. NO. 41EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV A DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL A SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMT DNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL M SDGSFFL ASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG. Pertuzumab VH WT, CH1 14.3.1.2A(K145A S188G), N297Q, 7.8.60A SEQ. ID. NO. 42EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV A DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL G SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMT DNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL M SDGSFFL ASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG. Pertuzumab VL WT, CK WTSEQ. ID. NO. 43 DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.Pertuzumab VL WT, CK 14.1.2 (E123K S131R) SEQ. ID. NO. 44DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSD K QLKSGTA R VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.Pertuzumab VL WT, CK 14.3.1.1A and 14.3.1.2A (S131R S176I)SEQ. ID. NO. 45 DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA R VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL I STLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.pEHGI Matuzumab VH WT, CH1 WT, N297Q, 7.8.60B SEQ. ID. NO. 46QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVRQAPGQGLEWIGEFNPSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDGRYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR R P R VYTLPPSREEMTKNQVSLV CLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYS VLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G.Matuzumab VH WT, CH1 14.3.1.1B and 14.3.1.2B (K221E), N297Q, 7.8.60BSEQ. ID. NO. 47 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVRQAPGQGLEWIGEFNPSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDGRYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVD EKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR R P R VYTLPPSREEMTKNQVSLV CLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYS VLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G. Matuzumab VL WT, CK WTSEQ. ID. NO. 48 DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGKAPKLLIYDTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC.Matuzumab VL WT, CK 14.3.1.1B and 14.3.1.2B (E123K) SEQ. ID. NO. 49DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGKAPKLLIYDTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTFGQG TKVEIKRTVAAPSVFIFPPSDK QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC.BHA10 VH WT, CH1 WT, N297Q, 7.8.60B SEQ. ID. NO. 50QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR R P R VYTL PPSREEMTKNQVSL VCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYS VLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG. BHA10 VH WT, CH1 14.3.1.1B and14.3.1.2B (K221E), N297Q 7.8.60B SEQ. ID. NO. 51QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVD EKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR R P R VYTL PPSREEMTKNQVSL VCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYS VLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG. BHA10 VL WT, CK WTSEQ. ID. NO. 52 DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.BHA10 VL WT, CK 14.3.1.1B and 14.3.1.2B (E123K) SEQ. ID. NO. 53DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKRTVAAPSVFIFPPSD K QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.WT IgG1_V(-) SEQ. ID. NO. 54:ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDK GSIEGRGSTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG WT CKappa_V(-) SEQ. ID. NO. 55:RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC14.1.2A IgG1_V(-) SEQ. ID. NO. 56: ASTKGPSVFPLAPSSKSTSGGTAALGCLV ADYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD EKVEP KSCDK GSIEGRGS THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 14.1.2a CKappa SEQ. ID. NO. 57:RTVAAPSVFIFPPSD K QLKSGTA R VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC14.3.1.1A IgG1_V(-) SEQ. ID. NO. 58: ASTKGPSVFPLAPSSKSTSGGTAALGCLV ADYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSL ASVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDK GSIEGRGSTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 14.3.1.1a and 14.3.1.2a CKappaSEQ. ID. NO. 59: RTVAAPSVFIFPPSDEQLKSGTARVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLISTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC14.3.1.1B and 14.3.1.2B IgG1_V(-) SEQ. ID. NO. 60:ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD E KVEP KSCDK GSIEGRGSTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 14.3.1.1b and 14.3.1.2b CKappaSEQ. ID. NO. 61: RTVAAPSVFIFPPSD K QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC14.3.1.2A IgG1_V(-) SEQ ID NO: 62ASTKGPSVFPLAPSSKSTSGGTAALGCLVADYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLGSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKGSIEGRGSTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 15.1A IgG1_V(-) SEQ. ID. NO. 63:ASTKGPSVFPLAPSSKSTSGGTAALGCLV A DYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSL ASVVTVPSSSLGTQTYICNVNHKPSNTKVD E KVEP KSCDK GSIEGRGSTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 15.1a and 15.2a CKappaSEQ. ID. NO. 64: RTVAAPSVFIFPPSD K QLKSGTA R VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL I STLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC15.2A IgG1_V(-) SEQ. ID. NO. 65: ASTKGPSVFPLAPSSKSTSGGTAALGCLV ADYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSL G SVVTVPSSSLGTQTYICNVNHKPSNTKVD EKVEP KSCDK GSIEGRGS THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG WT IgG1_CH1 SEQ. ID. NO. 66:ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV 14.1.2A IgG1_CH1SEQ. ID. NO. 67: ASTKGPSVFPLAPSSKSTSGGTAALGCLV A DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD E KV 14.3.1.1A IgG1_CH1SEQ. ID. NO. 68: ASTKGPSVFPLAPSSKSTSGGTAALGCLV A DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL A SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV14.3.1.1B and 14.3.1.2B IgG1_CH1 SEQ. ID. NO. 69:ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD E KV 14.3.1.2A IgG1_CH1SEQ. ID. NO: 70 ASTKGPSVFPLAPSSKSTSGGTAALGCLVADYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLGSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV 15.1A IgG1_CH1SEQ. ID. NO. 71: ASTKGPSVFPLAPSSKSTSGGTAALGCLV A DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL A SVVTVPSSSLGTQTYICNVNHKPSNTKVD E KV 15.2A IgG1_CH1SEQ. ID. NO. 72: ASTKGPSVFPLAPSSKSTSGGTAALGCLV A DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL G SVVTVPSSSLGTQTYICNVNHKPSNTKVD E KV Human IgG1 Fc_(WT)SEQ. ID. NO. 73 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK.Human IgG1 Fc_(7.8_A CH3) SEQ. ID. NO. 74APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVL MSDGSFFL A SKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK.Human IgG1 Fc_(7.8.60_A CH3) SEQ. ID. NO. 75APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT D NQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLM SDGSFFL A SKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK.Human IgG1 Fc_(20.8_A, 20.8.31_A or 20.8.33_A CH3) SEQ. ID. NO. 76APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQV STLPPSREEMTKNQVSL V CLV Y GFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYS VLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK.Human IgG1 Fc_(20.8.26_A, 20.8.34_A or 20.8.37_A CH3) SEQ. ID. NO. 77APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQV STLPPSREEMTKNQVSL M CLV Y GFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYS VLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK.Human IgG1 Fc_(7.8_B or 7.4_B CH3) SEQ. ID. NO. 78APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL V CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS V LTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK.Human IgG1 Fc_(7.8.60_B CH3) SEQ. ID. NO. 79APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPR R P RVYTLPPSREEMTKNQVSL V CLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYS VLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK.Human IgG1 Fc_(20.8_B or 20.8.26_B CH3) SEQ. ID. NO. 80APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE D MTKNQV Q LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL A SKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK.Human IgG1 Fc_(20.8.33_B or 20.8.34_B CH3) SEQ. ID. NO. 81APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR G D MTKNQV Q LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL A SKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK.Human IgG1 Fc_(20.8.31_B or 20.8.37_B CH3) SEQ. ID. NO. 82APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE D MTKNQV R LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL A SKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK.Human IgG1 Fc_(7.4_A CH3) SEQ. ID. NO. 83APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL A SKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK.Human IgG1 Fc_(7.4_B+366M CH3) SEQ. ID. NO. 84APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL M CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS V LTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK.(Bold underlined residues represent mutations toparental mAb or WT sequence)

We claim:
 1. A first and second fragment, antigen binding (Fab)comprising: a first heavy chain variable domain (V_(H)), a first humanIgG heavy chain constant (C_(H)1) domain having an alanine at residue145 according to Kabat Numbering and an alanine or a glycine at residue188 according to Kabat Numbering, a first light chain variable (V_(L))domain, and a first human light chain kappa constant (Ck) domain havingan arginine at residue 131 according to Kabat Numbering and anisoleucine at residue 176 according to Kabat Numbering; and a secondheavy chain variable (V_(H)) domain, a second human constant C_(H)1domain, a second light chain variable (V_(L)) domain, and a second humanlight chain kappa (Ck) domain, wherein each of the first V_(H) domainand the first V_(L) domain comprise three complementarity determiningregions (CDRs) which direct binding to a first antigen, each of thesecond V_(H) domain and the second V_(L) domain comprise three CDRswhich direct binding to a second antigen that differs from the firstantigen, and (i) the second human C_(H)1 domain has the wild-type humanIgG C_(H)1 sequence and the second human Ck domain has the wild typehuman Ck sequence, (ii) the second human C_(H)1 domain comprises anisoleucine at residue 188 according to Kabat Numbering and the secondhuman Ck domain has an alanine or a glycine at residue 176 according toKabat Numbering, or (iii) the second human C_(H)1 domain comprises aglutamic acid at residue 221 according to Kabat Numbering and the secondhuman Ck domain has lysine at residue 123 according to Kabat Numbering.2. The first and second fragment, antigen binding (Fab) according toclaim 1, wherein the first human C_(H)1 domain further comprises aglutamic acid at residue 221 according to Kabat Numbering and the humanCk domain further comprises a lysine at residue 123 according to KabatNumbering and wherein the second human C_(H)1 domain has the wild-typehuman IgG C_(H)1 sequence and the second human Ck domain has thewild-type human Ck sequence.
 3. The first and second fragment, antigenbinding (Fab) according to claim 1, wherein (a) the first V_(H) domaincomprises a glutamic acid at residue 62 and a lysine at residue 39according to Kabat Numbering, (b) the first V_(L) domain is kappaisotype and comprises an arginine at residue 1 and an aspartic acid atresidue 38 according to Kabat Numbering, (c) the second V_(H) domaincomprises a tyrosine at residue 39 and an arginine at residue 105according to Kabat Numbering, and (d) the second V_(L) domain is kappaisotype and comprises an arginine at residue 38 and an aspartic acid atresidue 42 according to Kabat Numbering.
 4. The first and secondfragment, antigen binding (Fab) according to claim 1, wherein (a) thefirst V_(H) domain comprises a tyrosine at residue 39 and an arginine atresidue 105 according to Kabat Numbering, (b) the first V_(L) domain iskappa isotype and comprises an arginine at residue 38 and an asparticacid at residue 42 according to Kabat Numbering, (c) the second V_(H)domain comprises a glutamic acid at residue 62 and a lysine at residue39 according to Kabat Numbering, and (d) the second V_(L) domain iskappa isotype and comprises an arginine at residue 1 and an asparticacid at residue 38 according to Kabat Numbering.
 5. The first and secondfragment, antigen binding (Fab) according to claim 1, wherein each ofthe first and second human C_(H)1 domains are individually IgG1 or IgG4isotype.
 6. The first and second fragment, antigen binding (Fab)according to claim 5, wherein each of the first and second human C_(H)1domains are IgG1 isotype.
 7. The first and second fragment, antigenbinding (Fab) according to claim 5, wherein each of the first and secondhuman C_(H)1 domains are IgG4 isotype.
 8. An IgG bispecific antibodycomprising: a first Fab comprising a first V_(H) domain, a first humanIgG constant region comprising a first human C_(H)1 domain having analanine at residue 145 according to Kabat Numbering and an alanine or aglycine at residue 188 according to Kabat Numbering, a first V_(L)domain, and a first human Ck domain comprising an arginine at residue131 according to Kabat Numbering and an isoleucine at residue 176according to Kabat Numbering; and a second Fab comprising a second V_(H)domain, a second human V_(L) domain, and a human IgG constant regioncomprising a second human C_(H)1 domain and a second human Ck domain,wherein each of the first V_(H) domain and the first V_(L) domaincomprise three CDRs which direct binding to a first antigen, each of thesecond heavy chain and light chain variable domains comprise three CDRswhich direct binding to a second antigen that differs from the firstantigen, and (i) the second human C_(H)1 domain having a wild-type humanIgG C_(H)1 sequence and the second human Ck domain having a wild typehuman Ck sequence, (ii) the second human C_(H)1 domain comprises anisoleucine at residue 188 according to Kabat Numbering and the secondhuman Ck domain has an alanine or a glycine at residue 176 according toKabat Numbering, or (iii) the second human IgG heavy chain constantregion comprises a second human C_(H)1 domain that comprises a glutamicacid at residue 221 according to Kabat Numbering and the second human Ckdomain has lysine at residue 123 according to Kabat Numbering.
 9. TheIgG bispecific antibody according to claim 8, wherein the first humanC_(H)1 domain further comprises glutamic acid at residue 221 accordingto Kabat Numbering and the first human Ck domain further comprises alysine at residue 123 according to Kabat Numbering, wherein the secondhuman C_(H)1 domain has the wild-type human IgG C_(H)1 sequence and thesecond human Ck domain has the wild-type human Ck sequence.
 10. The IgGbispecific antibody according to claim 8, wherein (a) the first V_(H)domain comprises a glutamic acid at residue 62 and a lysine at residue39 according to Kabat Numbering, (b) the first V_(L) domain is kappaisotype and comprises an arginine at residue 1 and an aspartic acid atresidue 38 according to Kabat Numbering, (c) the second V_(H) domaincomprises a tyrosine at residue 39 and an arginine at residue 105according to Kabat Numbering, and (d) the second V_(L) domain is kappaisotype and comprises an arginine at residue 38 and an aspartic acid atresidue 42 according to Kabat Numbering.
 11. The IgG bispecific antibodyaccording to claim 8, wherein (a) the first V_(H) domain comprises atyrosine at residue 39 and an arginine at residue 105 according to KabatNumbering, (b) the first V_(L) domain is kappa isotype and comprises anarginine at residue 38 and an aspartic acid at residue 42 according toKabat Numbering; (c) the second V_(H) domain comprises a glutamic acidat residue 62 and a lysine at residue 39 according to Kabat Numbering,and (d) the second V_(L) domain is kappa isotype and comprises anarginine at residue 1 and an aspartic acid at residue 38 according toKabat Numbering.
 12. The IgG bispecific antibody according to claim 8,wherein one of the first or second human IgG constant regions comprisesa CH3 domain comprising an alanine at residue 407 according to EU IndexNumbering; and the other of the first or second human IgG constantregions comprises a CH3 domain comprising a valine or methionine atresidue 366 and a valine at residue 409 according to EU Index Numbering.13. The IgG bispecific antibody according to claim 8, wherein one of thefirst or second human IgG constant regions comprises a C_(H)3 domainhaving an alanine at residue 407, a methionine at residue 399, and anaspartic acid at residue 360 according to EU Index Numbering; and theother of the first or second human IgG constant regions comprising a CH3domain having a valine at residue 366, a valine at residue 409, and anarginine at residues 345 and 347 according to EU Index Numbering. 14.The IgG bispecific antibody according to claim 8, wherein one of thefirst or second human IgG constant regions comprises a C_(H)3 domainhaving an alanine at residue 407, a glycine at residue 356, an asparticacid at residue 357, and a glutamine at residue 364 according to EUIndex Numbering; and the other of the first or second human IgG constantregions comprises a C_(H)3 domain having a methionine at residue 366, avaline at residue 409, a serine at residue 349, and a tyrosine atresidue 370 according to EU Index Numbering.
 15. The IgG bispecificantibody according to claim 8, wherein each of the first and secondhuman C_(H)1 domains are IgG1 or IgG4 isotype.
 16. The IgG bispecificantibody according to claim 15, wherein each of the first human C_(H)1domain and the second human C_(H)1 domain are IgG1 isotype.
 17. The IgGbispecific antibody according to claim 15, wherein each of the firsthuman C_(H)1 domain and the second human C_(H)1 domain are IgG4 isotype.18. The IgG bispecific antibody according to claim 8, wherein each ofthe first V_(L) domain and the second V_(L) domain is human kappaisotype.
 19. A first and second fragment, antigen binding (Fab)comprising: (a) a first heavy chain variable domain (V_(H)) and a firsthuman IgG heavy chain constant C_(H)1 (C_(H)1) domain having an alanineat residue 145 according to Kabat Numbering and a glutamic acid atresidue 221 according to Kabat Numbering; (b) a first light chainvariable domain (V_(L)) and a first human light chain kappa constant(Ck) domain having an arginine at residue 131 according to KabatNumbering and a lysine at residue 123 according to Kabat Numbering; (c)a second heavy chain variable domain V_(H) and a second human IgG heavychain constant C_(H)1 domain having a wild-type human IgG C_(H)1sequence; and (d) a second light chain variable V_(L) domain and asecond human light chain kappa constant Ck domain having a wild typehuman Ck sequence, wherein each of the first V_(H) domain and the V_(L)domain comprise three complementarity determining regions (CDRs) whichdirect binding to a first antigen, and each of the second V_(H) andV_(L) domains comprise three CDRs which direct binding to a secondantigen that differs from the first antigen.
 20. The first and secondfragment, antigen binding (Fab) of claim 19, wherein, (a) the firstV_(H) domain comprises a glutamic acid at residue 62 and a lysine atresidue 39 according to Kabat Numbering, (b) the first V_(L) domain iskappa isotype and comprises an arginine at residue 1 and an asparticacid at residue 38 according to Kabat Numbering, (c) the second V_(H)domain comprises a tyrosine at residue 39 and an arginine at residue 105according to Kabat Numbering, and (d) the second V_(L) domain is kappaisotype and comprises an arginine at residue 38 and an aspartic acid atresidue 42 according to Kabat Numbering.
 21. The first and secondfragment, antigen binding (Fab) according to claim 19, wherein (a) thefirst V_(H) domain comprises a tyrosine at residue 39 and an arginine atresidue 105 according to Kabat Numbering, (b) the first V_(L) domain iskappa isotype and comprises an arginine at residue 38 and an asparticacid at residue 42 according to Kabat Numbering, (c) the second V_(H)domain comprises a glutamic acid at residue 62 and a lysine at residue39 according to Kabat Numbering, and (d) the second V_(L) domain iskappa isotype and comprises an arginine at residue 1 and an asparticacid at residue 38 according to Kabat Numbering.
 22. The first andsecond fragment, antigen binding (Fab) according to claim 19, whereineach of the first human C_(H)1 domain and the second human C_(H)1 domainare individually IgG1 or IgG4 isotype.
 23. The first and secondfragment, antigen binding (Fab) of claim 22, wherein each of the firsthuman C_(H)1 domain and the second human C_(H)1 domain are IgG1 isotype.24. The first and second fragment, antigen binding (Fab) of claim 22,wherein each of the first human C_(H)1 domain and the second humanC_(H)1 domain are IgG4 isotype.
 25. A bispecific antibody comprising: afirst Fab comprising a first V_(H) domain, a first V_(L) domain, a firsthuman IgG constant region comprising a first human C_(H)1 domain havingan alanine at residue 145 according to Kabat Numbering and an alanineand a glutamic acid at residue 221 according to Kabat Numbering, and afirst human Ck domain comprising an arginine at residue 131 according toKabat Numbering and a lysine at residue 123 according to KabatNumbering; and a second Fab comprising a second V_(H) domain, a secondV_(L) domain, and a human IgG constant region comprising a second humanC_(H)1 domain having a wild-type human IgG C_(H)1 sequence and a secondhuman Ck domain having a wild type human Ck sequence, wherein each ofthe first V_(H) domain and the first V_(L) domain comprise three CDRswhich direct binding to a first antigen, and each of the second heavychain variable domain and the second light chain variable domaincomprise three CDRs which direct binding to a second antigen thatdiffers from the first antigen.
 26. A bispecific antibody comprisingaccording to claim 25, wherein, (a) the first V_(H) domain comprises aglutamic acid at residue 62 and a lysine at residue 39 according toKabat Numbering, (b) the first V_(L) domain is kappa isotype andcomprises an arginine at residue 1 and an aspartic acid at residue 38according to Kabat Numbering, (c) the second V_(H) domain comprises atyrosine at residue 39 and an arginine at residue 105 according to KabatNumbering, and (d) the second V_(L) domain is kappa isotype andcomprises an arginine at residue 38 and an aspartic acid at residue 42according to Kabat Numbering.
 27. The bispecific antibody according toclaim 25, wherein (a) the first V_(H) domain comprises a tyrosine atresidue 39 and an arginine at residue 105 according to Kabat Numbering,(b) the first V_(L) domain is kappa isotype and comprises an arginine atresidue 38 and an aspartic acid at residue 42 according to KabatNumbering, (c) the second V_(H) domain comprises a glutamic acid atresidue 62 and a lysine at residue 39 according to Kabat Numbering, and(d) the second V_(L) domain is kappa isotype and comprises an arginineat residue 1 and an aspartic acid at residue 38 according to KabatNumbering.
 28. The bispecific antibody according to claim 25, whereinone of the first human IgG constant region or the second human IgGconstant region comprises a C_(H)3 domain having an alanine at residue407 with residue according to EU Index Numbering; and the other of thefirst human IgG constant region or the second human IgG constant regioncomprises a CH3 domain having a valine or methionine at residue 366 anda valine at residue 409 according to EU Index Numbering.
 29. Thebispecific antibody according to claim 25, wherein one of the firsthuman IgG constant region or the second human IgG constant regioncomprises a C_(H)3 domain having an alanine at residue 407, a methionineat residue 399, and an aspartic acid at residue 360 according to EUIndex Numbering; and the other of the first human IgG constant region orthe second human IgG constant region comprises a C_(H)3 domain having avaline at residue 366, a valine at residue 409, and an arginine atresidues 345 and 347 according to EU Index Numbering.
 30. The bispecificantibody according to claim 25, wherein one of the first human IgGconstant region or the second human IgG constant region comprises aC_(H)3 domain having an alanine at residue 407, a glycine at residue356, an aspartic acid at residue 357, and a glutamine at residue 364according to EU Index Numbering; and the other of the first human IgGconstant region or the second human IgG constant region comprises aC_(H)3 domain having a methionine at residue 366, a valine at residue409, a serine at residue 349, and a tyrosine at residue 370 according toEU Index Numbering.
 31. The bispecific antibody according to claim 25,wherein each of the first human C_(H)1 domain and the second humanC_(H)1 domain are IgG1 or IgG4 isotype.
 32. The bispecific antibodyaccording to claim 31, wherein each of the first human C_(H)1 domain andthe second human C_(H)1 domains are IgG1 isotype.
 33. The bispecificantibody according to claim 31, wherein each of the first human C_(H)1domain and the second human C_(H)1 domain are IgG4 isotype.
 34. Thebispecific antibody according to claim 25, wherein each of the firstV_(L) domain and the second V_(L) domain is human kappa isotype.